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Sample records for active site functional

  1. 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.

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

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  3. Synergistic effect between defect sites and functional groups on the hydrolysis of cellulose over activated carbon.

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups.

  4. Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase.

    PubMed

    Collman, James P; Decréau, Richard A

    2008-11-07

    A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).

  5. Molybdenum and tungsten oxygen transferases--and functional diversity within a common active site motif.

    PubMed

    Pushie, M Jake; Cotelesage, Julien J; George, Graham N

    2014-01-01

    Molybdenum and tungsten are the only second and third-row transition elements with a known function in living organisms. The molybdenum and tungsten enzymes show common structural features, with the metal being bound by a pyranopterin-dithiolene cofactor called molybdopterin. They catalyze a variety of oxygen transferase reactions coupled with two-electron redox chemistry in which the metal cycles between the +6 and +4 oxidation states usually with water, either product or substrate, providing the oxygen. The functional roles filled by the molybdenum and tungsten enzymes are diverse; for example, they play essential roles in microbial respiration, in the uptake of nitrogen in green plants, and in human health. Together, the enzymes form a superfamily which is among the most prevalent known, being found in all kingdoms of life. This review discusses what is known of the active site structures and the mechanisms, together with some recent insights into the evolution of these important enzyme systems.

  6. 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

  7. Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

    PubMed Central

    2012-01-01

    Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance) of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs), which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites. PMID:22536854

  8. Noncovalent intermolecular interactions between dehydroepiandrosterone and the active site of human dehydroepiandrosterone sulphotransferase: A density functional theory based treatment

    NASA Astrophysics Data System (ADS)

    Astani, Elahe; Heshmati, Emran; Chen, Chun-Jung; Hadipour, Nasser L.; Shekarsaraei, Setareh

    2016-04-01

    A theoretical study was performed to characterize noncovalent intermolecular interactions, especially hydrogen bond (HB), in the active site of enzyme human dehydroepiandrosterone sulphotransferase (SULT2A1/DHEA) using the local (M06-L) and hybrid (M06, M06-2X) meta-GGA functionals of density functional theory (DFT). Results revealed that DHEA is able to form HBs with residues His99, Tyr231, Met137 and Met16 in the active site of the SULT2A1/DHEA. It was found that DHEA interacts with the other residues through electrostatic and Van der Waals interactions.

  9. AutoDock-GIST: Incorporating Thermodynamics of Active-Site Water into Scoring Function for Accurate Protein-Ligand Docking.

    PubMed

    Uehara, Shota; Tanaka, Shigenori

    2016-11-23

    Water plays a significant role in the binding process between protein and ligand. However, the thermodynamics of water molecules are often underestimated, or even ignored, in protein-ligand docking. Usually, the free energies of active-site water molecules are substantially different from those of waters in the bulk region. The binding of a ligand to a protein causes a displacement of these waters from an active site to bulk, and this displacement process substantially contributes to the free energy change of protein-ligand binding. The free energy of active-site water molecules can be calculated by grid inhomogeneous solvation theory (GIST), using molecular dynamics (MD) and the trajectory of a target protein and water molecules. Here, we show a case study of the combination of GIST and a docking program and discuss the effectiveness of the displacing gain of unfavorable water in protein-ligand docking. We combined the GIST-based desolvation function with the scoring function of AutoDock4, which is called AutoDock-GIST. The proposed scoring function was assessed employing 51 ligands of coagulation factor Xa (FXa), and results showed that both scoring accuracy and docking success rate were improved. We also evaluated virtual screening performance of AutoDock-GIST using FXa ligands in the directory of useful decoys-enhanced (DUD-E), thus finding that the displacing gain of unfavorable water is effective for a successful docking campaign.

  10. Elucidating the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts: A density functional theory study

    NASA Astrophysics Data System (ADS)

    Wang, Ziyun; Liu, Xinyi; Rooney, D. W.; Hu, P.

    2015-10-01

    The dehydrogenation of cyclohexanol to cyclohexanone is very important in the manufacture of nylon. Copper-based catalysts are the most popular catalysts for this reaction, and on these catalysts the reaction mechanism and active site are in debate. In order to elucidate the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts, density functional theory with dispersion corrections were performed on up to six facets of copper in two different oxidation states: monovalent copper and metallic copper. By calculating the surface energies of these facets, Cu(111) and Cu2O(111) were found to be the most stable facets for metallic copper and for monovalent copper, respectively. On these two facets, all the possible elementary steps in the dehydrogenation pathway of cyclohexanol were calculated, including the adsorption, dehydrogenation, hydrogen coupling and desorption. Two different reaction pathways for dehydrogenation were considered on both surfaces. It was revealed that the dehydrogenation mechanisms are different on these two surfaces: on Cu(111) the hydrogen belonging to the hydroxyl is removed first, then the hydrogen belonging to the carbon is subtracted, while on Cu2O(111) the hydrogen belonging to the carbon is removed followed by the subtraction of the hydrogen in the hydroxyl group. Furthermore, by comparing the energy profiles of these two surfaces, Cu2O(111) was found to be more active for cyclohexanol dehydrogenation than Cu(111). In addition, we found that the coordinatively unsaturated copper sites on Cu2O(111) are the reaction sites for all the steps. Therefore, the coordinatively unsaturated copper site on Cu2O(111) is likely to be the active site for cyclohexanol dehydrogenation on the copper-based catalysts.

  11. Crystal Structures and Functional Characterization of Wild Type and Active Sites Mutants of CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Poulos, Thomas L.

    2014-01-01

    Although CYP101D1 and P450cam catayze the same reaction at a similar rate and share strikingly similar active site architectures, there are significance functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what can differ yet maintain high catalytic efficiency. Crystal structures of the cyanide complex of wild type CYP101D1 and it active site mutants, D259N and T260A, have been solved. The conformational changes in CYP101D1 upon cyanide binding are very similar to P450cam indicating a similar mechanism for proton delivery during oxygen activation using solvent assisted proton transfer. The D259N-CN− complex shows a perturbed solvent structure compared to wild type which is similar to what was observed in the oxy-complex of the corresonding D251N mutant in P450cam. As in P450cam the T260A mutant is highly uncoupled while the D259N gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provides a strucutral basis for understanding these functional differences. PMID:24261604

  12. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  13. Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

    PubMed

    Qureshi, Shabir H; Yang, Likui; Manithody, Chandrashekhara; Bae, Jong-Sup; Rezaie, Alireza R

    2008-09-01

    Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

  14. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  15. SitesIdentify: a protein functional site prediction tool

    PubMed Central

    2009-01-01

    Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify), based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/ PMID:19922660

  16. The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.

    PubMed

    Martinez, Salette; Wu, Rui; Sanishvili, Ruslan; Liu, Dali; Holz, Richard

    2014-01-29

    Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp(2)) form to a tetrahedral form (sp(3)). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase-boronic acid complexes represent a "snapshot" of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys(113)-OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.

  17. Simultaneous presence of both open metal sites and free functional organic sites in a noncentrosymmetric dynamic metal-organic framework with bimodal catalytic and sensing activities.

    PubMed

    Saha, Rajat; Joarder, Biplab; Roy, Anupam Singha; Manirul Islam, Sk; Kumar, Sanjay

    2013-12-02

    Assimilation of open metal sites (OMSs) and free functional organic sites (FOSs) with a framework strut has opened up a new route for the fabrication of novel metal-organic materials, thereby providing a unique opportunity to explore their multiple functionalities. A new metal-organic framework (MOF), {[Cu(ina)2(H2O)][Cu(ina)2(bipy)]·2H2O}n (1) (ina=isonicotinate, bipy=4,4'-bipyridine), has been synthesized and characterized. Complex 1 is crystallized in the orthorhombic noncentrosymmetric space group Aba2 and consists of two different 2D coordination polymers, [Cu(ina)2(H2O)]n and [Cu(ina)2(bipy)]n, with entrapped solvent water molecules. Hydrogen-bonding interactions assemble these two different 2D coordination layers in a single-crystal structure with interdigitation of pendant 4,4'-bipy from one layer into the groove of another. Upon removal of guest molecules, 1 undergoes a structural transformation in single-crystal-to-single-crystal fashion with expansion of the effective void space. Each metal center is five-coordinated and thus can potentially behave as an OMS, and the free pyridyl groups of pendant 4,4'-bipy moieties and free -C=O groups can act as free FOSs. Thus, owing to presence of both OMSs and free FOSs, the framework exhibits multifunctional properties. Owing to the presence of OMSs, the framework can act as a Lewis acid catalyst as well as a small-molecule sensor material, and in a similar way, owing to the presence of free FOSs, it performs as a Lewis base catalyst and a cation sensor material. Furthermore, owing to noncentrosymmetry with large polarity along a particular direction, it shows strong second-harmonic generation/nonlinear optical (SHG-NLO) activity.

  18. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.

    PubMed

    Zhang, Huijun; Guo, Wenbin; Xu, Changan; Zhou, Hongbo; Chen, Xinhua

    2013-12-14

    Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

  19. Linking structure to function: The search for active sites in non-platinum group metal oxygen reduction reaction catalysts

    SciTech Connect

    Holby, Edward F.; Zelenay, Piotr

    2016-05-17

    Atomic-scale structures of oxygen reduction reaction (ORR) active sites in non-platinum group metal (non-PGM) catalysts, made from pyrolysis of carbon, nitrogen, and transition-metal (TM) precursors have been the subject of continuing discussion in the fuel cell electrocatalysis research community. We found that quantum chemical modeling is a path forward for understanding of these materials and how they catalyze the ORR. Here, we demonstrate through literature examples of how such modeling can be used to better understand non-PGM ORR active site relative stability and activity and how such efforts can also aid in the interpretation of experimental signatures produced by these materials.

  20. The pathway for serial proton supply to the active site of nitrogenase: enhanced density functional modeling of the Grotthuss mechanism.

    PubMed

    Dance, Ian

    2015-11-07

    Nitrogenase contains a well defined and conserved chain of water molecules leading to the FeMo cofactor (FeMo-co, an [Fe7MoCS9] cluster with bidentate chelation of Mo by homocitrate) that is the active site where N2 and other substrates are sequentially hydrogenated using multiple protons and electrons. The function of this chain is proposed to be a proton wire, serially translocating protons to triply-bridging S3B of FeMo-co, where, concomitant with electron transfer to FeMo-co, an H atom is generated on S3B. Density functional simulations of this proton translocation mechanism are reported here, using a large 269-atom model that includes all residues hydrogen bonded to and surrounding the water chain, and likely to influence proton transfer: three carboxylate O atoms of obligatory homocitrate are essential. The mechanism involves the standard two components of the Grotthuss mechanism, namely H atom slides that shift H3O(+) from one water site to the next, and HOH molecular rotations that convert backward (posterior) OH bonds in the water chain to forward (anterior) OH bonds. The topography of the potential energy surface for each of these steps has been mapped. H atom slides pass through very short (ca. 2.5 Å) O-H-O hydrogen bonds, while HOH rotations involve the breaking of O-HO hydrogen bonds, and the occurrence of long (up to 3.6 Å) separations between contiguous water molecules. Both steps involve low potential energy barriers, <7 kcal mol(-1). During operation of the Grotthuss mechanism in nitrogenase there are substantial displacements of water molecules along the chain, occurring as ripples. These characteristics of the 'Grotthuss two-step', coupled with a buffering ability of two carboxylate O atoms of homocitrate, and combined with density functional characterisation of the final proton slide from the ultimate water molecule to S3B (including electron addition), have been choreographed into a complete mechanism for serial hydrogenation of FeMo-co. The

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

    PubMed

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

    2016-05-17

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

  2. Density functional theory study of model complexes for the revised nitrate reductase active site in Desulfovibrio desulfuricans NapA.

    PubMed

    Hofmann, Matthias

    2009-09-01

    [Mo(SSCH3)(S2C2(CH3)2)2](x) complexes with charges x between -3 and +3 were investigated by density functional theory computations as minimal nitrate reductase active-site models. The strongly reduced species (x = -2, -3) exist preferentially as pentacoordinate sulfo complexes separated from a thiolate anion. The oxidized extremes (x > 0) clearly prefer hexacoordinate complexes with an eta(2)-MeSS ligand. Among the neutral and especially for the singly negatively charged species structures with eta(2)-MeSS and eta(1)-MeSS ligands are energetically close to the sulfo methyl sulfide complex without SS bonding. For x = -1 the three isomers lie in a 1.5 kcal mol(-1) energy range. Putative mechanistic pathways for nitrate reduction from the literature were investigated computationally: (1) reduction at a pentacoordinate sulfo complex, (2) reduction at the ligand, and (3) reduction at the molybdenum center with an R-S-S ligand. All three pathways could be traced at least for some overall charges but no definite conclusion can be drawn about the mechanism. Complexes with larger dithiolato ligands were also computed in order to model the tricyclic metallopterin framework more accurately: the first heterocyclus (5,6-dihydro-2H-pyran) stabilizes the nitrate complex and the molybdenum oxo product complex by approximately 10 kcal mol(-1) and also reduces the activation barrier (by approximately 5 kcal mol(-1)). The effect of the second (1,2,3,4-tetrahydropyrazin) and third heterocyclus (2-amino-3H-pyrimidin-4-one) on the relative energies is relatively small. For bigger models derived from an experimental protein structure, nitrate reduction at a persulfo molybdenum(IV) complex fragment (mechanism 3) is clearly favored over the oxidation of a molybdenum-bound sulfur atom (mechanism 2). Mechanism 1 could not be investigated for the big models but seems the least favorable on the basis of the results from smaller models.

  3. KatB, a cyanobacterial Mn-catalase with unique active site configuration: Implications for enzyme function.

    PubMed

    Bihani, Subhash C; Chakravarty, Dhiman; Ballal, Anand

    2016-04-01

    Manganese catalases (Mn-catalases), a class of H2O2 detoxifying proteins, are structurally and mechanistically distinct from the commonly occurring catalases, which contain heme. Active site of Mn-catalases can serve as template for the synthesis of catalase mimetics for therapeutic intervention in oxidative stress related disorders. However, unlike the heme catalases, structural aspects of Mn-catalases remain inadequately explored. The genome of the ancient cyanobacterium Anabaena PCC7120, shows the presence of two Mn-catalases, KatA and KatB. Here, we report the biochemical and structural characterization of KatB. The KatB protein (with a C-terminal his-tag) was over-expressed in Escherichia coli and purified by affinity chromatography. On the addition of Mn(2+) to the E. coli growth medium, a substantial increase in production of the soluble KatB protein was observed. The purified KatB protein was an efficient catalase, which was relatively insensitive to inhibition by azide. Crystal structure of KatB showed a hexameric assembly with four-helix bundle fold, characteristic of the Ferritin-like superfamily. With canonical Glu4His2 coordination geometry and two terminal water ligands, the KatB active site was distinctly different from that of other Mn-catalases. Interestingly, the KatB active site closely resembled the active sites of ruberythrin/bacterioferritin, bi-iron members of the Ferritin-like superfamily. The KatB crystal structure provided fundamental insights into the evolutionary relationship within the Ferritin-like superfamily and further showed that Mn-catalases can be sub-divided into two groups, each with a distinct active site configuration.

  4. Linking structure to function: The search for active sites in non-platinum group metal oxygen reduction reaction catalysts

    DOE PAGES

    Holby, Edward F.; Zelenay, Piotr

    2016-05-17

    Atomic-scale structures of oxygen reduction reaction (ORR) active sites in non-platinum group metal (non-PGM) catalysts, made from pyrolysis of carbon, nitrogen, and transition-metal (TM) precursors have been the subject of continuing discussion in the fuel cell electrocatalysis research community. We found that quantum chemical modeling is a path forward for understanding of these materials and how they catalyze the ORR. Here, we demonstrate through literature examples of how such modeling can be used to better understand non-PGM ORR active site relative stability and activity and how such efforts can also aid in the interpretation of experimental signatures produced by thesemore » materials.« less

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

    PubMed Central

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

    2016-01-01

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

  6. An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins

    PubMed Central

    Babbitt, Patricia C.; Ferrin, Thomas E.

    2017-01-01

    Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially—MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method’s novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated

  7. An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

    PubMed Central

    Knutson, Stacy T.; Westwood, Brian M.; Leuthaeuser, Janelle B.; Turner, Brandon E.; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D.; Harper, Angela F.; Brown, Shoshana D.; Morris, John H.; Ferrin, Thomas E.; Babbitt, Patricia C.

    2017-01-01

    Abstract Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. PMID:28054422

  8. An approach to functionally relevant clustering of the protein universe: Active site profile-based clustering of protein structures and sequences.

    PubMed

    Knutson, Stacy T; Westwood, Brian M; Leuthaeuser, Janelle B; Turner, Brandon E; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D; Harper, Angela F; Brown, Shoshana D; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C; Fetrow, Jacquelyn S

    2017-04-01

    Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification-amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two-Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure-Function Linkage Database, SFLD) self-identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self-identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well-curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP-identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F-measure and performance analysis on the enolase search results and comparison to GEMMA and SCI-PHY demonstrate that TuLIP avoids the over-division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.

  9. Amplified Rate Acceleration by Simultaneous Up-Regulation of Multiple Active Sites in an Endo-Functionalized Porous Capsule.

    PubMed

    Kopilevich, Sivil; Müller, Achim; Weinstock, Ira A

    2015-10-14

    Using the hydrolysis of epoxides in water as a model reaction, the effect of multiple active sites on Michaelis-Menten compliant rate accelerations in a porous capsule is demonstrated. The capsule is a water-soluble Ih-symmetry Keplerate-type complex of the form, [{Mo(VI)6O21(H2O)6}12{Mo(V)2O4(L)}30](42-), in which 12 pentagonal "ligands," {(Mo(VI))Mo(VI)5O21(H2O)6}(6-), are coordinated to 30 dimolybdenum sites, {Mo(V)2O4L}(1+) (L = an endohedrally coordinated η(2)-bound carboxylate anion), resulting in 20 Mo9O9 pores. When "up-regulated" by removal of ca. one-third of the blocking ligands, L, an equal number of dimolybdenum sites are activated, and the newly freed-up space allows for encapsulation of nearly twice as many substrate guests, leading to a larger effective molarity (amplification), and an increase in the rate acceleration (k(cat)/k(uncat)) from 16,000 to an enzyme-like value of 182,800.

  10. Functional analyses for tRNase Z variants: an aspartate and a histidine in the active site are essential for the catalytic activity.

    PubMed

    Elbarbary, Reyad A; Takaku, Hiroaki; Nashimoto, Masayuki

    2008-12-01

    We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.

  11. Functional activation independently contributes to naming ability and relates to lesion site in post-stroke aphasia.

    PubMed

    Skipper-Kallal, Laura M; Lacey, Elizabeth H; Xing, Shihui; Turkeltaub, Peter E

    2017-04-01

    Language network reorganization in aphasia may depend on the degree of damage in critical language areas, making it difficult to determine how reorganization impacts performance. Prior studies on remapping of function in aphasia have not accounted for the location of the lesion relative to critical language areas. They rectified this problem by using a multimodal approach, combining multivariate lesion-symptom mapping and fMRI in chronic aphasia to understand the independent contributions to naming performance of the lesion and the activity in both hemispheres. Activity was examined during two stages of naming: covert retrieval, and overt articulation. Regions of interest were drawn based on over- and under-activation, and in areas where activity had a bivariate relationship with naming. Regressions then tested whether activation of these regions predicted naming ability, while controlling for lesion size and damage in critical left hemisphere naming areas, as determined by lesion-symptom mapping. Engagement of the right superior temporal sulcus (STS) and disengagement of the left dorsal pars opercularis (dPOp) during overt naming was associated with better than predicted naming performance. Lesions in the left STS prevented right STS engagement and resulted in persistent left dPOp activation. In summary, changes in activity during overt articulation independently relate to naming outcomes, controlling for stroke severity. Successful remapping relates to network disruptions that depend on the location of the lesion in the left hemisphere. Hum Brain Mapp 38:2051-2066, 2017. © 2017 Wiley Periodicals, Inc.

  12. Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme.

    PubMed

    Knap, A K; Pratt, R F

    1991-01-01

    The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence [Knap & Pratt (1989) Proteins Struct. Funct. Genet. 6, 316-323] for the activation of the active-site thiol group by adjacent functional groups. The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site. The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1. It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234). A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated. A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.

  13. New insights into the catalytic mechanism of histidine phosphatases revealed by a functionally essential arginine residue within the active site of the Sts phosphatases.

    PubMed

    San Luis, Boris; Nassar, Nicolas; Carpino, Nick

    2013-07-01

    Sts (suppressor of T-cell receptor signalling)-1 and Sts-2 are HPs (histidine phosphatases) that negatively regulate TCR (T-cell receptor) signalling pathways, including those involved in cytokine production. HPs play key roles in such varied biological processes as metabolism, development and intracellular signalling. They differ considerably in their primary sequence and substrate specificity, but possess a catalytic core formed by an invariant quartet of active-site residues. Two histidine and two arginine residues cluster together within the HP active site and are thought to participate in a two-step dephosphorylation reaction. To date there has been little insight into any additional residues that might play an important functional role. In the present study, we identify and characterize an additional residue within the Sts phosphatases (Sts-1 Arg383 or Sts-2 Arg369) that is critical for catalytic activity and intracellular function. Mutation of Sts-1 Arg383 to an alanine residue compromises the enzyme's activity and renders Sts-1 unable to suppress TCR-induced cytokine induction. Of the multiple amino acids substituted for Arg383, only lysine partially rescues the catalytic activity of Sts-1. Although Sts-1 Arg383 is conserved in all Sts homologues, it is only conserved in one of the two sub-branches of HPs. The results of the present study highlight an essential role for Sts-1 phosphatase activity in regulating T-cell activation and add a new dimension of complexity to our understanding of HP catalytic activity.

  14. Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

    PubMed Central

    Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

    2016-01-01

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  15. Invited award contribution for ACS Award in Inorganic Chemistry. Geometric and electronic structure contributions to function in bioinorganic chemistry: active sites in non-heme iron enzymes.

    PubMed

    Solomon, E I

    2001-07-16

    Spectroscopy has played a major role in the definition of structure/function correlations in bioinorganic chemistry. The importance of spectroscopy combined with electronic structure calculations is clearly demonstrated by the non-heme iron enzymes. Many members of this large class of enzymes activate dioxygen using a ferrous active site that has generally been difficult to study with most spectroscopic methods. A new spectroscopic methodology has been developed utilizing variable temperature, variable field magnetic circular dichroism, which enables one to obtain detailed insight into the geometric and electronic structure of the non-heme ferrous active site and probe its reaction mechanism on a molecular level. This spectroscopic methodology is presented and applied to a number of key mononuclear non-heme iron enzymes leading to a general mechanistic strategy for O2 activation. These studies are then extended to consider the new features present in the binuclear non-heme iron enzymes and applied to understand (1) the mechanism of the two electron/coupled proton transfer to dioxygen binding to a single iron center in hemerythrin and (2) structure/function correlations over the oxygen-activating enzymes stearoyl-ACP Delta9-desaturase, ribonucleotide reductase, and methane monooxygenase. Electronic structure/reactivity correlations for O2 activation by non-heme relative to heme iron enzymes will also be developed.

  16. Identification of key functional residues in the active site of human {beta}1,4-galactosyltransferase 7: a major enzyme in the glycosaminoglycan synthesis pathway.

    PubMed

    Talhaoui, Ibtissam; Bui, Catherine; Oriol, Rafael; Mulliert, Guillermo; Gulberti, Sandrine; Netter, Patrick; Coughtrie, Michael W H; Ouzzine, Mohamed; Fournel-Gigleux, Sylvie

    2010-11-26

    Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.

  17. Redox-active on-surface polymerization of single-site divalent cations from pure metals by a ketone-functionalized phenanthroline

    SciTech Connect

    Skomski, Daniel; Tempas, Christopher D.; Bukowski, Gregory S.; Smith, Kevin A.; Tait, Steven L.

    2015-03-14

    Metallic iron, chromium, or platinum mixing with a ketone-functionalized phenanthroline ligand on a single crystal gold surface demonstrates redox activity to a well-defined oxidation state and assembly into thermally stable, one dimensional, polymeric chains. The diverging ligand geometry incorporates redox-active sub-units and bi-dentate binding sites. The gold surface provides a stable adsorption environment and directs growth of the polymeric chains, but is inert with regard to the redox chemistry. These systems are characterized by scanning tunnelling microscopy, non-contact atomic force microscopy, and X-ray photoelectron spectroscopy under ultra-high vacuum conditions. The relative propensity of the metals to interact with the ketone group is examined, and it is found that Fe and Cr more readily complex the ligand than Pt. The formation and stabilization of well-defined transition metal single-sites at surfaces may open new routes to achieve higher selectivity in heterogeneous catalysts.

  18. Calcium-manganese oxides as structural and functional models for active site in oxygen evolving complex in photosystem II: lessons from simple models.

    PubMed

    Najafpour, Mohammad Mahdi

    2011-01-01

    The oxygen evolving complex in photosystem II which induces the oxidation of water to dioxygen in plants, algae and certain bacteria contains a cluster of one calcium and four manganese ions. It serves as a model to split water by sunlight. Reports on the mechanism and structure of photosystem II provide a more detailed architecture of the oxygen evolving complex and the surrounding amino acids. One challenge in this field is the development of artificial model compounds to study oxygen evolution reaction outside the complicated environment of the enzyme. Calcium-manganese oxides as structural and functional models for the active site of photosystem II are explained and reviewed in this paper. Because of related structures of these calcium-manganese oxides and the catalytic centers of active site of the oxygen evolving complex of photosystem II, the study may help to understand more about mechanism of oxygen evolution by the oxygen evolving complex of photosystem II.

  19. Redox-active on-surface polymerization of single-site divalent cations from pure metals by a ketone-functionalized phenanthroline

    NASA Astrophysics Data System (ADS)

    Skomski, Daniel; Tempas, Christopher D.; Bukowski, Gregory S.; Smith, Kevin A.; Tait, Steven L.

    2015-03-01

    Metallic iron, chromium, or platinum mixing with a ketone-functionalized phenanthroline ligand on a single crystal gold surface demonstrates redox activity to a well-defined oxidation state and assembly into thermally stable, one dimensional, polymeric chains. The diverging ligand geometry incorporates redox-active sub-units and bi-dentate binding sites. The gold surface provides a stable adsorption environment and directs growth of the polymeric chains, but is inert with regard to the redox chemistry. These systems are characterized by scanning tunnelling microscopy, non-contact atomic force microscopy, and X-ray photoelectron spectroscopy under ultra-high vacuum conditions. The relative propensity of the metals to interact with the ketone group is examined, and it is found that Fe and Cr more readily complex the ligand than Pt. The formation and stabilization of well-defined transition metal single-sites at surfaces may open new routes to achieve higher selectivity in heterogeneous catalysts.

  20. Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site.

    PubMed Central

    Ma, L; Weeda, G; Jochemsen, A G; Bootsma, D; Hoeijmakers, J H; van der Eb, A J

    1992-01-01

    The human XPBC/ERCC-3 gene, which corrects the excision-repair defect in xeroderma pigmentosum group B cells and the UV-sensitive CHO mutant 27-1 cells, appears to be expressed constitutively in various cell types and tissues. We have analysed the structure and functionality of the XPBC/ERCC-3 promoter. Transcription of the XPBC/ERCC-3 gene is initiated from heterogeneous sites, with a major startpoint mapped at position -54 (relative to the translation start codon ATG). The promoter region does not possess classical TATA and CAAT elements, but it is GC-rich and contains three putative Sp1-binding sites. In addition, there are two elements related to the cyclic AMP (cAMP)-response element (CRE) and the 12-O-tetradecanoyl phorbol-13-acetate-response element (TRE) in the 5'-flanking region. Transient expression analysis of XPBC/ERCC-3 promoter-CAT chimeric plasmids revealed that a 127-bp fragment, spanning position -129 to -3, is minimally required for the promoter activity. Transcription of the XPBC/ERCC-3 promoter depends on the integrity of a putative Sp1-binding site in close proximity to the major cap site. Band shift assays showed that this putative Sp1-binding site can interact specifically with a nuclear factor, most likely transcription factor Sp1 (or an Sp1-like factor) in vitro. Images PMID:1741247

  1. Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site.

    PubMed

    Hegeman, A D; Gross, J W; Frey, P A

    2001-06-05

    A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ). From the model, two groups of active site residues were identified. The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH). These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are

  2. On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase.

    PubMed

    Montero-Morán, G M; Lara-González, S; Alvarez-Añorve, L I; Plumbridge, J A; Calcagno, M L

    2001-08-28

    side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.

  3. 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.

  4. The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

    PubMed Central

    Hunninghake, G W; Monks, B G; Geist, L J; Monick, M M; Monroy, M A; Stinski, M F; Webb, A C; Dayer, J M; Auron, P E; Fenton, M J

    1992-01-01

    Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation. Images PMID:1630455

  5. Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

    PubMed Central

    Lednicky, J; Folk, W R

    1992-01-01

    The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

  6. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  7. Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups.

    PubMed

    Knap, A K; Pratt, R F

    1989-01-01

    The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc. Natl. Acad. Sci. U.S.A. 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group. The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid. beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group).

  8. Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

    PubMed

    Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-03-25

    Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology and designing enzymes with new functional capabilities. This article is protected by copyright. All rights reserved.

  9. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

    PubMed Central

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  10. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    NASA Astrophysics Data System (ADS)

    Murphy, Jesse; Mullins, Elwood; Kappock, T.

    2016-05-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates less than 3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analogue dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  11. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    DOE PAGES

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-05-23

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes andmore » orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

  12. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    SciTech Connect

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-05-23

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  13. Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase.

    PubMed

    Reschke, Stefan; Niks, Dimitri; Wilson, Heather; Sigfridsson, Kajsa G V; Haumann, Michael; Rajagopalan, K V; Hille, Russ; Leimkühler, Silke

    2013-11-19

    Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (kcat/KM) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt). X-ray absorption spectroscopy revealed a Mo(VI)-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O2Mo(VI)S2(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD4Ser was altered compared to those of SOMD4Ser and SOMD4Cys(wt), with g1 in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.

  14. 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.

  15. A Casein Kinase II Phosphorylation Site in AtYY1 Affects Its Activity, Stability, and Function in the ABA Response

    PubMed Central

    Wu, Xiu-Yun; Li, Tian

    2017-01-01

    The phosphorylation and dephosphorylation of proteins are crucial in the regulation of protein activity and stability in various signaling pathways. In this study, we identified an ABA repressor, Arabidopsis Ying Yang 1 (AtYY1) as a potential target of casein kinase II (CKII). AtYY1 physically interacts with two regulatory subunits of CKII, CKB3, and CKB4. Moreover, AtYY1 can be phosphorylated by CKII in vitro, and the S284 site is the major CKII phosphorylation site. Further analyses indicated that S284 phosphorylation can enhance the transcriptional activity and protein stability of AtYY1 and hence strengthen the effect of AtYY1 as a negative regulator in the ABA response. Our study provides novel insights into the regulatory mechanism of AtYY1 mediated by CKII phosphorylation. PMID:28348572

  16. Requirements for functional models of the iron hydrogenase active site: D2/H2O exchange activity in ((mu-SMe)(mu-pdt)[Fe(CO)2(PMe3)]2+)[BF4-].

    PubMed

    Georgakaki, Irene P; Miller, Matthew L; Darensbourg, Marcetta Y

    2003-04-21

    Hydrogen uptake in hydrogenase enzymes can be assayed by H/D exchange reactivity in H(2)/D(2)O or H(2)/D(2)/H(2)O mixtures. Diiron(I) complexes that serve as structural models for the active site of iron hydrogenase are not active in such isotope scrambling but serve as precursors to Fe(II)Fe(II) complexes that are functional models of [Fe]H(2)ase. Using the same experimental protocol as used previously for ((mu-H)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-H(+) (Zhao et al. J. Am. Chem. Soc. 2001, 123, 9710), we now report the results of studies of ((mu-SMe)(mu-pdt)[Fe(CO)(2)(PMe(3))](2)(+)), 1-SMe(+), toward H/D exchange. The 1-SMe(+) complex can take up H(2) and catalyze the H/D exchange reaction in D(2)/H(2)O mixtures under photolytic, CO-loss conditions. Unlike 1-H(+), it does not catalyze H(2)/D(2) scrambling under anhydrous conditions. The molecular structure of 1-SMe(+) involves an elongated Fe.Fe separation, 3.11 A, relative to 2.58 A in 1-H(+). It is proposed that the strong SMe(-) bridging ligand results in catalytic activity localized on a single Fe(II) center, a scenario that is also a prominent possibility for the enzyme active site. The single requirement is an open site on Fe(II) available for binding of D(2) (or H(2)), followed by deprotonation by the external base H(2)O (or D(2)O).

  17. Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.

    PubMed Central

    Meng, M.; Chane, T. L.; Sun, Y. J.; Hsiao, C. D.

    1999-01-01

    Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis. PMID:10595547

  18. A study of hydrogen bond effects on the oxygen, nitrogen, and hydrogen electric field gradient tensors in the active site of human dehydroepiandrosterone sulphotransferase: A density-functional theory based treatment

    NASA Astrophysics Data System (ADS)

    Astani, Elahe; Heshmati, Emran; Chen, Chun-Jung; Hadipour, Nasser L.; Shekarsaraei, Setareh

    2016-06-01

    An investigation of the density functional theory (DFT) was carried out to study the effects of the hydrogen bond (HB) interactions on 2H, 14N, and 17O electric field gradient (EFG) tensors in the active site of human dehydroepiandrosterone sulphotransferase (SULT2A1/DHEA). Natural Bonding Orbital (NBO) analysis was also performed on this active site. The results revealed that the magnitude of the quadrupole coupling constant (QCC) change at each nucleus directly depends on its contribution amount to the HB interaction.

  19. Lanthanide Metal-Organic Frameworks with Six-Coordinated Ln(III) Ions and Free Functional Organic Sites for Adsorptions and Extensive Catalytic Activities

    PubMed Central

    Zhu, Yu; Zhu, Min; Xia, Li; Wu, Yunlong; Hua, Hui; Xie, Jimin

    2016-01-01

    Three chelating-amino-functionalized lanthanide metal-organic frameworks, Y-DDQ, Dy-DDQ and Eu-DDQ, were synthesized with a flexible dicarboxylate ligand based on quinoxaline (H2DDQ = N, N′-dibenzoic acid-2,3-diaminoquinoxaline). The three-dimensional framework is constructed by the H2DDQ linkers connecting the zigzag ladders, showing a net of sra topology. In the structures, one kind of Ln(III) ions metal centers are six-coordinated and thus can potentially behave as open metal sites (OMSs), while the free chelating amino groups can act as free functional organic sites (FOSs). The N2 and Ar adsorption behaviors indicate that these Ln-DDQ exhibits stable microporous frameworks with high surface area after remove of the solvents. Owing to presence of OMSs and FOSs, these MOFs show good ability of CO2, dyes captures and Lewis acid catalyst for cyanosilylation reaction. In view of the existing FOSs in the framework, Pd NPs were immobilized onto the MOFs through graft interactions between free chelating amino groups and metal ions precursor using postsynthetic modification. The well dispersed Pd@Ln-DDQs exhibit efficient and recyclable catalytic reduction of 4-nitrophenol to 4-aminophenol, and they can also act as an excellent catalyst for Suzuki-Miyaura cross-coupling reactions with the exposed Pd NPs. PMID:27431731

  20. Lanthanide Metal-Organic Frameworks with Six-Coordinated Ln(III) Ions and Free Functional Organic Sites for Adsorptions and Extensive Catalytic Activities

    NASA Astrophysics Data System (ADS)

    Zhu, Yu; Zhu, Min; Xia, Li; Wu, Yunlong; Hua, Hui; Xie, Jimin

    2016-07-01

    Three chelating-amino-functionalized lanthanide metal-organic frameworks, Y-DDQ, Dy-DDQ and Eu-DDQ, were synthesized with a flexible dicarboxylate ligand based on quinoxaline (H2DDQ = N, N‧-dibenzoic acid-2,3-diaminoquinoxaline). The three-dimensional framework is constructed by the H2DDQ linkers connecting the zigzag ladders, showing a net of sra topology. In the structures, one kind of Ln(III) ions metal centers are six-coordinated and thus can potentially behave as open metal sites (OMSs), while the free chelating amino groups can act as free functional organic sites (FOSs). The N2 and Ar adsorption behaviors indicate that these Ln-DDQ exhibits stable microporous frameworks with high surface area after remove of the solvents. Owing to presence of OMSs and FOSs, these MOFs show good ability of CO2, dyes captures and Lewis acid catalyst for cyanosilylation reaction. In view of the existing FOSs in the framework, Pd NPs were immobilized onto the MOFs through graft interactions between free chelating amino groups and metal ions precursor using postsynthetic modification. The well dispersed Pd@Ln-DDQs exhibit efficient and recyclable catalytic reduction of 4-nitrophenol to 4-aminophenol, and they can also act as an excellent catalyst for Suzuki-Miyaura cross-coupling reactions with the exposed Pd NPs.

  1. Structure and function relationship of toxin from Chinese scorpion Buthus martensii Karsch (BmKAGAP): gaining insight into related sites of analgesic activity.

    PubMed

    Cui, Yong; Guo, Gui-Li; Ma, Lin; Hu, Nan; Song, Yong-Bo; Liu, Yan-Feng; Wu, Chun-Fu; Zhang, Jing-Hai

    2010-06-01

    In this study, an effective Escherichia coli expression system was used to study the role of residues in the antitumor-analgesic peptide from Chinese scorpion Buthus martensii Karsch (BmKAGAP). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, nine mutants of BmKAGAP were obtained by PCR. Using site-directed mutagenesis, all of these residues were individually substituted by one amino acid. These were then subjected to a circular dichroism analysis, and an analgesic activity assay in mice. This study represents a thorough mapping and elucidation of the epitopes that underlie the molecular basis of the analgesic activity. The three-dimensional structure of BmKAGAP was established by homology modeling. Our results revealed large mutant-dependent differences that indicated important roles for the studied residues. With our ongoing efforts for establishing the structure and analgesic activity relationship of BmKAGAP, we have succeeded in pinpointing which residues are important for the analgesic activity.

  2. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  3. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  4. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  5. Extra-gonadal sites of estrogen biosynthesis and function

    PubMed Central

    Barakat, Radwa; Oakley, Oliver; Kim, Heehyen; Jin, Jooyoung; Ko, CheMyong Jay

    2016-01-01

    Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496] PMID:27530684

  6. Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.

    PubMed

    Sakaguchi, Masayoshi; Matsushima, Yudai; Nagamine, Yusuke; Matsuhashi, Tomoki; Honda, Shotaro; Okuda, Shoi; Ohno, Misa; Sugahara, Yasusato; Shin, Yongchol; Oyama, Fumitaka; Kawakita, Masao

    2017-03-01

    Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

  7. HANFORD SITE WELDING PROGRAM SUCCESSFULLY PROVIDING A SINGLE SITE FUNCTION FOR USE BY MULTIPLE CONTRACTORS

    SciTech Connect

    CANNELL GR

    2009-11-19

    The Department of Energy, Richland Operations (DOE-RL) recently restructured its Hanford work scope, awarding two new contracts over the past several months for a total of three contracts to manage the sites cleanup efforts. DOE-RL met with key contractor personnel prior to and during contract transition to ensure site welding activities had appropriate oversight and maintained code compliance. The transition also provided an opportunity to establish a single site-wide function that would provide welding and materials engineering services to the Hanford site contractors: CH2M HILL Plateau Remediation Company (CHPRC); Mission Support Alliance (MSA); Washington River Protection Solutions (WRPS); and Washington Closure Hanford (WCH). Over the years, multiple and separate welding programs (amongst the several contractors) existed at the Hanford site leading to inefficiencies resulting from duplication of administrative efforts, maintenance of welding procedures, welder performance certifications, etc. The new, single program eliminates these inefficiencies. The new program, co-managed by two of the sites' new contractors, the CHPRC ('owner' of the program and responsible for construction welding services) and the MSA (provides maintenance welding services), provides more than just the traditional construction and maintenance welding services. Also provided, are welding engineering, specialty welding development/qualification for the closure of radioactive materials containers and materials evaluation/failure analysis. The following describes the new Hanford site welding program.

  8. Patterns of oligonucleotide distribution within DNA and RNA functional sites

    SciTech Connect

    Kolchanov, N.A.; Kel, A.E.; Ponomarenko, M.P.; Romachenko, A.G.; Likchachev, J.; Milanesi, L.; Lim, H.

    1993-12-31

    Patterns of short oligonucleotide distribution within DNA and RNA functional sites have been analyzed using ``Site-Video`` computer system. The group of DNA functional sites involved nucleosome binding sites, gyrase cleavage sites, promoters of E. coli and men. The group of RNA functional sites involved donor and acceptor splice sites of men, translation initiation sites of E. coli and men and translation frame shift site sites. Analysis of these samples of nucleotide sequences have been carried out by the ``Site-Video`` computer system. For each type of site specific set of patterns of oligonucleotide distribution important for the functioning and recognition have been revealed. At the same time, the number of specific patterns revealed in RNA sites was significantly higher than those in DNA sites. On the base of the results obtained, the script of functional sites for evolutionary emergency have been prompted. According to it, two types of context feature selection took place: (1) positive selection targeted to the appearance of the definite types of context features in particular regions of functional sites;and (2) negative selection targeted to the elimination of definite types of context features in particular regions of functional sites. The authors suppose that evolutionary formation of any functional site is a multistep process realized via combination of positive and negative selections. Negative selection, via fixation of a specific pattern of mutations, eliminates false signals of regulatory proteins binding with the functional site. Positive selection leads to the appearance of local context features (signals) which provide for the specificity and efficiency of the site functioning.

  9. High Precision Prediction of Functional Sites in Protein Structures

    PubMed Central

    Buturovic, Ljubomir; Wong, Mike; Tang, Grace W.; Altman, Russ B.; Petkovic, Dragutin

    2014-01-01

    We address the problem of assigning biological function to solved protein structures. Computational tools play a critical role in identifying potential active sites and informing screening decisions for further lab analysis. A critical parameter in the practical application of computational methods is the precision, or positive predictive value. Precision measures the level of confidence the user should have in a particular computed functional assignment. Low precision annotations lead to futile laboratory investigations and waste scarce research resources. In this paper we describe an advanced version of the protein function annotation system FEATURE, which achieved 99% precision and average recall of 95% across 20 representative functional sites. The system uses a Support Vector Machine classifier operating on the microenvironment of physicochemical features around an amino acid. We also compared performance of our method with state-of-the-art sequence-level annotator Pfam in terms of precision, recall and localization. To our knowledge, no other functional site annotator has been rigorously evaluated against these key criteria. The software and predictive models are incorporated into the WebFEATURE service at http://feature.stanford.edu/wf4.0-beta. PMID:24632601

  10. [Natural organic compounds that affect to microtubule functions: syntheses and structure-activity relationships of combretastatins, curacin A and their analogs as the colchicine-site ligands on tubulin].

    PubMed

    Iwasaki, S; Shirai, R

    2000-10-01

    Microtubules (MT) are cylindrical polymers of the protein tubulin (TN) alpha, beta-heterodimer, and are known to be the main component of spindles in mitotic apparatus of eucaryotic cells. MT are also involved in many other basic and essential cell functions. There are a number of natural and synthetic compounds that interfere with MT function to cause the mitotic arrest of eucaryotic cells. Such antimitotic agents show a broad biological activity, and can be used for medicinal and agrochemical purposes. On the other hand, they are important also as the biochemical tools for understanding the dynamics of MT network. Most of such antimitotic agents, with a few exceptions, bind to beta-TN. Among them, colchicine (CLC), vinblastine and taxol have played major roles in practical uses as well as in biochemical studies of MT functions. They all bind to beta-TN but their binding sites are different. We have worked on a variety of antimitotic agents that bind to either of colchicine-site, vinblastine-site and taxol-site, in discovery, structures, biological actions and/or interactions with TN. In this paper, the results of our studies on CLC-site ligands were summarized; (1) synthetic analogs of combretastatin A-4 (CBS A-4), isolated as a cytotoxic compound produced by a species of South African tree Combretum caffrum, (2) curacin A (CU-A), a cytotoxic metabolite of a marine cyanobacteria Lyngbya majuscula, and its related compounds. Interactions of these compounds with TN were studied and structure-activity relationships of these two classes of compounds were discussed.

  11. Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes

    PubMed Central

    Jack, Benjamin R.; Meyer, Austin G.; Echave, Julian; Wilke, Claus O.

    2016-01-01

    Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes. PMID:27138088

  12. Text Mining Improves Prediction of Protein Functional Sites

    PubMed Central

    Cohn, Judith D.; Ravikumar, Komandur E.

    2012-01-01

    We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

  13. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  14. Plasmodium subtilisin-like protease 1 (SUB1): Insights into the active-site structure, specificity and function of a pan-malaria drug target

    PubMed Central

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J.

    2012-01-01

    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop ‘pan-reactive’ drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. PMID:22543039

  15. Biomimetic oxidation studies. 9. Mechanistic aspects of the oxidation of alcohols with functional,active site methane monooxygenase enzyme models in aqueous solution

    SciTech Connect

    Rabion, A. ||; Chen, S.; Wang, J.; Buchanan, R.M.; Seris, J.L.; Fish, R.H. |

    1995-12-13

    The syntheses of biomimetic complexes that mimic the major structural features of the hydroxylase component of methane monooxygenase enzyme (MMO) and, more importantly, that provide similar alkane functionalization activity, in the presence of an oxidant, have been of great interest to the discipline of bioinorganic chemistry. In this communication, we will demonstrate the feasibility of conducting biomimetic oxidation studies in H{sub 2}O with soluble substrates, i.e., alcohols (cyclohexanol, benzyl alcohol), using H{sub 2}O-stable MMO mimics at pH 4.2, and the oxidant, tert-butyl hydroperoxide (TBHP). Both the Mitusunobu procedure and the mesylate displacement reaction proceeded with complete inversion of the stereo-center and provided optically pure penultimate intermediate (>99.9% ee). The synthesis was completed by reduction of the nitro group under standard conditions to deliver LY300164 in 87%. In summary, we have developed an efficient and environmentally benign synthesis of the 5H-2,3-benzodiazepine LY300164 that provides the optically pure compound in 51% overall yield. Intramolecular hydrazone alkylation led to a remarkably facile and selective formation of the benzodiazepine. Furthermore, the application of resins to whole-cell-based biotransformations should find general utility for similar reactions that are complicated by component inhibition and product isolation. 11 refs., 1 fig.

  16. 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.

  17. 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.

  18. Density functional theory calculations on the active site of biotin synthase: mechanism of S transfer from the Fe(2)S(2) cluster and the role of 1st and 2nd sphere residues.

    PubMed

    Rana, Atanu; Dey, Subal; Agrawal, Amita; Dey, Abhishek

    2015-10-01

    Density functional theory (DFT) calculations are performed on the active site of biotin synthase (BS) to investigate the sulfur transfer from the Fe(2)S(2) cluster to dethiobiotin (DTB). The active site is modeled to include both the 1st and 2nd sphere residues. Molecular orbital theory considerations and calculation on smaller models indicate that only an S atom (not S²⁻) transfer from an oxidized Fe(2)S(2) cluster leads to the formation of biotin from the DTB using two adenosyl radicals generated from S-adenosyl-L-methionine. The calculations on larger protein active site model indicate that a 9-monothiobiotin bound reduced cluster should be an intermediate during the S atom insertion from the Fe(2)S(2) cluster consistent with experimental data. The Arg260 bound to Fe1, being a weaker donor than cysteine bound to Fe(2), determines the geometry and the electronic structure of this intermediate. The formation of this intermediate containing the C9-S bond is estimated to have a ΔG(≠) of 17.1 kcal/mol while its decay by the formation of the 2nd C6-S bond is calculated to have a ΔG(≠) of 29.8 kcal/mol, i.e. the 2nd C-S bond formation is calculated to be the rate determining step in the cycle and it leads to the decay of the Fe(2)S(2) cluster. Significant configuration interaction (CI), present in these transition states, helps lower the barrier of these reactions by ~30-25 kcal/mol relative to a hypothetical outer-sphere reaction. The conserved Phe285 residue near the Fe(2)S(2) active site determines the stereo selectivity at the C6 center of this radical coupling reaction. Reaction mechanism of BS investigated using DFT calculations. Strong CI and the Phe285 residue control the kinetic rate and stereochemistry of the product.

  19. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  20. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  1. Functional constraints on adaptive evolution of protein ubiquitination sites

    PubMed Central

    Lu, Liang; Li, Yang; Liu, Zhongyang; Liang, Fengji; Guo, Feifei; Yang, Shuai; Wang, Dan; He, Yangzhige; Xiong, Jianghui; Li, Dong; He, Fuchu

    2017-01-01

    It is still unclear whether there exist functional constraints on the evolution of protein ubiquitination sites, because most previous studies regarded all protein ubiquitination sites as a whole or only focused on limited structural properties. We tried to clarify the relation between functional constraints and ubiquitination sites evolution. We investigated the evolutionary conservation of human ubiquitination sites in a broad evolutionary scale from G. gorilla to S. pombe, and we found that in organisms originated after the divergence of vertebrate, ubiquitination sites are more conserved than their flanking regions, while the opposite tendency is observed before this divergence time. By grouping the ubiquitination proteins into different functional categories, we confirm that many functional constraints like certain molecular functions, protein tissue expression specificity and protein connectivity in protein-protein interaction network enhance the evolutionary conservation of ubiquitination sites. Furthermore, by analyzing the gains of ubiquitination sites at different divergence time and their functional characters, we validate that the emergences of ubiquitination sites at different evolutionary time were also affected by the uncovered functional constraints. The above results suggest that functional constraints on the adaptive evolution of ubiquitination sites increase the opportunity for ubiquitination to synthetically regulate various cellular and developmental processes during evolution. PMID:28054638

  2. Localizing the site of magnetic brain stimulation by functional MRI.

    PubMed

    Terao, Y; Ugawa, Y; Sakai, K; Miyauchi, S; Fukuda, H; Sasaki, Y; Takino, R; Hanajima, R; Furubayashi, T; Pütz, B; Kanazawa, I

    1998-07-01

    In order to locate the site of action of transcranial magnetic stimulation (TMS) within the human motor cortices, we investigated how the optimal positions for evoking motor responses over the scalp corresponded to the hand and leg primary-motor areas. TMS was delivered with a figure-8 shaped coil over each point of a grid system constructed on the skull surface, each separated by 1 cm, to find the optimal site for obtaining motor-evoked potentials (MEPs) in the contralateral first dorsal interosseous (FDI) and tibialis anterior (TA) muscles. Magnetic resonance imaging scans of the brain were taken for each subject with markers placed over these sites, the positions of which were projected onto the cortical region just beneath. On the other hand, cortical areas where blood flow increased during finger tapping or leg movements were identified on functional magnetic resonance images (fMRI), which should include the hand and leg primary-motor areas. The optimal location for eliciting MEPs in FDI, regardless of their latency, lay just above the bank of the precentral gyrus, which coincided with the activated region during finger tapping in fMRI studies. The direction of induced current preferentially eliciting MEPs with the shortest latency in each subject was nearly perpendicular to the course of the precentral gyrus at this position. The optimal site for evoking motor responses in TA was also located just above the activated area during leg movements identified within the anterior portion of the paracentral lobule. The results suggest that, for magnetic stimulation, activation occurs in the primary hand and leg motor area (Brodmann area 4), which is closest in distance to the optimal scalp position for evoking motor responses.

  3. The single site Green s function and Krein s theorem

    SciTech Connect

    Wang, Yang; Stocks, George Malcolm; Faulkner, John Sam

    2014-01-01

    An important step in electronic structure calculations using multiple-scattering theory is obtaining the density of states for the central site from the Green s function for that site. We have found that the Krein s spectral displacement function for the central site contributes significantly to the understanding of these calculations. We argue that these insights can lead to improvements in the robustness of MST electronic structure codes without negatively impacting their performance.

  4. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  5. 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.

  6. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  7. 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.

  8. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  9. Examining Search Functions of EAD Finding Aids Web Sites

    ERIC Educational Resources Information Center

    Zhou, Xiaomu

    2006-01-01

    This article examined the search functions for all individual EAD Web sites listed on the Library of Congress Web site in 2003. In particular, the type of search engine, search modes, options for searching, search results display, search feedback, and other features of the search systems were studied. The data analysis suggests that there have…

  10. Conserved Functional Motifs and Homology Modeling to Predict Hidden Moonlighting Functional Sites

    PubMed Central

    Wong, Aloysius; Gehring, Chris; Irving, Helen R.

    2015-01-01

    Moonlighting functional centers within proteins can provide them with hitherto unrecognized functions. Here, we review how hidden moonlighting functional centers, which we define as binding sites that have catalytic activity or regulate protein function in a novel manner, can be identified using targeted bioinformatic searches. Functional motifs used in such searches include amino acid residues that are conserved across species and many of which have been assigned functional roles based on experimental evidence. Molecules that were identified in this manner seeking cyclic mononucleotide cyclases in plants are used as examples. The strength of this computational approach is enhanced when good homology models can be developed to test the functionality of the predicted centers in silico, which, in turn, increases confidence in the ability of the identified candidates to perform the predicted functions. Computational characterization of moonlighting functional centers is not diagnostic for catalysis but serves as a rapid screening method, and highlights testable targets from a potentially large pool of candidates for subsequent in vitro and in vivo experiments required to confirm the functionality of the predicted moonlighting centers. PMID:26106597

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

    PubMed

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

    2013-08-14

    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.

  12. A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae.

    PubMed

    Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena; Davies, Christopher; Nicholas, Robert A

    2012-04-03

    Insertion of an aspartate residue at position 345a in penicillin-binding protein 2 (PBP 2), which lowers the rate of penicillin acylation by ~6-fold, is commonly observed in penicillin-resistant strains of Neisseria gonorrhoeae. Here, we show that insertions of other amino acids also lower the penicillin acylation rate of PBP 2, but none supported growth of N. gonorrhoeae, indicating loss of essential transpeptidase activity. The Asp345a mutation likely acts by altering the interaction between its adjacent residue, Asp346, in the β2a-β2d hairpin loop and Ser363, the middle residue of the SXN active site motif. Because the adjacent aspartate creates ambiguity in the position of the insertion, we also examined if insertions at position 346a could confer decreased susceptibility to penicillin. However, only aspartate insertions were identified, indicating that only an Asp-Asp couple can confer resistance and retain transpeptidase function. The importance of the Asp346-Ser363 interaction was assessed by mutation of each residue to Ala. Although both mutants lowered the acylation rate of penicillin G by 5-fold, neither could support growth of N. gonorrhoeae, again indicating loss of transpeptidase function. Interaction between a residue in the equivalent of the β2a-β2d hairpin loop and the middle residue of the SXN motif is observed in crystal structures of other Class B PBPs, and its importance is also supported by multisequence alignments. Overall, these results suggest that this conserved interaction can be manipulated (e.g., by insertion) to lower the acylation rate by β-lactam antibiotics and increase resistance, but only if essential transpeptidase activity is preserved.

  13. [Physical activity and brain function].

    PubMed

    Kempermann, G

    2012-06-01

    Physical activity has direct and indirect effects on brain function in health and disease. Findings demonstrating that physical activity improves cognitive and non-cognitive functions and is preventive for several neuropsychiatric disorders have attracted particular interest. This short review focuses on sports and physical exercise in normal brain function and summarizes which mechanisms might underlie the observed effects, which methodological problems exist, which relationships exist to concepts of plasticity and neural reserves and what evolutionary relevance the initially surprising finding that physical exercise is good for the brain has.

  14. Prediction of Functional Sites Based on the Fuzzy Oil Drop Model

    PubMed Central

    Bryliński, Michał; Prymula, Katarzyna; Jurkowski, Wiktor; Kochańczyk, Marek; Stawowczyk, Ewa; Konieczny, Leszek; Roterman, Irena

    2007-01-01

    A description of many biological processes requires knowledge of the 3-D structure of proteins and, in particular, the defined active site responsible for biological function. Many proteins, the genes of which have been identified as the result of human genome sequencing, and which were synthesized experimentally, await identification of their biological activity. Currently used methods do not always yield satisfactory results, and new algorithms need to be developed to recognize the localization of active sites in proteins. This paper describes a computational model that can be used to identify potential areas that are able to interact with other molecules (ligands, substrates, inhibitors, etc.). The model for active site recognition is based on the analysis of hydrophobicity distribution in protein molecules. It is shown, based on the analyses of proteins with known biological activity and of proteins of unknown function, that the region of significantly irregular hydrophobicity distribution in proteins appears to be function related. PMID:17530916

  15. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

    PubMed Central

    Bode, W.; Turk, D.; Karshikov, A.

    1992-01-01

    Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70

  16. 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.

  17. Structure-based Methods for Computational Protein Functional Site Prediction

    PubMed Central

    Dukka, B KC

    2013-01-01

    Due to the advent of high throughput sequencing techniques and structural genomic projects, the number of gene and protein sequences has been ever increasing. Computational methods to annotate these genes and proteins are even more indispensable. Proteins are important macromolecules and study of the function of proteins is an important problem in structural bioinformatics. This paper discusses a number of methods to predict protein functional site especially focusing on protein ligand binding site prediction. Initially, a short overview is presented on recent advances in methods for selection of homologous sequences. Furthermore, a few recent structural based approaches and sequence-and-structure based approaches for protein functional sites are discussed in details. PMID:24688745

  18. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2013-10-01

    mice and mice transfused with Syk inhibitor-treated platelets . Platelet lodging was remarkably decreased in lungs of mice transfused with Syk...AD_________________ Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet ...30September2012–29September2013 4. TITLE AND SUBTITLE Complement Activation Alters Platelet Function 5a. CONTRACT NUMBER W81XWH-12-1-0523 5b. GRANT NUMBER

  19. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  20. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

  1. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  2. Melanocortin MC₄ receptor expression sites and local function.

    PubMed

    Siljee-Wong, Jacqueline E

    2011-06-11

    The melanocortin MC(4) receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC(4) receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC(4) receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC(4) receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC(4) receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC(4) receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC(4) receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC(4) receptor at these sites.

  3. First Principles Computational Study of the Active Site of Arginase

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

    Ab initio density functional theory (DFT) methods were used to investigate the structural features of the active site of the binuclear enzyme rat liver arginase. Special emphasis was placed on the crucial role of the second shell ligand interactions. These interactions were systematically studied by performing calculations on models of varying size. It was determined that a water molecule, and not hydroxide, is the bridging exogenous ligand. The carboxylate ligands facilitate the close approach of the Mn (II) ions by attenuating the metal-metal electrostatic repulsion. Of the two metals, MnA was shown to carry a larger positive charge. Analysis of the electronic properties of the active site revealed that orbitals involving the terminal Asp234 residue, as well as the flexible -1,1 bridging Asp232, lie at high energies, suggesting weaker coordination. This is reflected in certain structural variability present in our models and is also consistent with recent experimental findings. Finally, implications of our findings for the biological function of the enzyme are delineated.

  4. An application of site response functions to ground motion prediction

    NASA Astrophysics Data System (ADS)

    Tsuda, K.; Archuleta, R.; Steidl, J.; Koketsu, K.

    2006-12-01

    The prediction of ground motion from large future earthquakes is very important for hazard mitigation in urban areas of Japan. Because the observed ground motions are affected by three factors; the seismic source, attenuation (quality factor) of seismic wave propagation inside the earth, and the effects of the local surface geology, understanding each factor is essential for the ground motion prediction. The effect of surface geology (local soil conditions) on ground motions was documented as early as the 1906 San Francisco earthquake. The correlation between soil type and the degree of damage was again recognized in the 1923 Kanto earthquake. Additionally, accelerometer records from almost all recent large events also have reinforced the role of site effects in the level of strong shaking. Because most cities in Japan are located on thick sedimentary basins, accounting for site response is essential for realistic predictions of ground motion. However, predicting ground motion has uncertainties that arise from all three factors: source, path, and site. The analysis of well-recorded data from dense seismograph arrays can reduce these uncertainties for ground motion prediction. The new technique presented here provides a site response correlation function for estimation of the spatial distribution of site response. This function is based on the known site responses at instrumented sites and is used to estimate the site response at a site for which there is no instrumental records. We initially predict the level of ground motion by using this estimate with the assumption of linear wave propagation. This method is applied to the data from a relatively dense seismic array located near the city of Sendai, Japan by using moderate sized earthquakes with small ground motion levels to estimate linear site response. The array consists of 29 stations: 20 managed by Tohoku Institute of Technology, 6 by Building Research Institute, and 3 by NIED within an area of 20 x 30 km

  5. 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.

  6. 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.

  7. 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.

  8. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  9. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

  10. Functional interaction of nitrogenous organic bases with cytochrome P450: a critical assessment and update of substrate features and predicted key active-site elements steering the access, binding, and orientation of amines.

    PubMed

    Hlavica, Peter

    2006-04-01

    The widespread use of nitrogenous organic bases as environmental chemicals, food additives, and clinically important drugs necessitates precise knowledge about the molecular principles governing biotransformation of this category of substrates. In this regard, analysis of the topological background of complex formation between amines and P450s, acting as major catalysts in C- and N-oxidative attack, is of paramount importance. Thus, progress in collaborative investigations, combining physico-chemical techniques with chemical-modification as well as genetic engineering experiments, enables substantiation of hypothetical work resulting from the design of pharmacophores or homology modelling of P450s. Based on a general, CYP2D6-related construct, the majority of prospective amine-docking residues was found to cluster near the distal heme face in the six known SRSs, made up by the highly variant helices B', F and G as well as the N-terminal portion of helix C and certain beta-structures. Most of the contact sites examined show a frequency of conservation < 20%, hinting at the requirement of some degree of conformational versatility, while a limited number of amino acids exhibiting a higher level of conservation reside close to the heme core. Some key determinants may have a dual role in amine binding and/or maintenance of protein integrity. Importantly, a series of non-SRS elements are likely to be operative via long-range effects. While hydrophobic mechanisms appear to dominate orientation of the nitrogenous compounds toward the iron-oxene species, polar residues seem to foster binding events through H-bonding or salt-bridge formation. Careful uncovering of structure-function relationships in amine-enzyme association together with recently developed unsupervised machine learning approaches will be helpful in both tailoring of novel amine-type drugs and early elimination of potentially toxic or mutagenic candidates. Also, chimeragenesis might serve in the construction

  11. 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

  12. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

  13. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  14. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

  15. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  16. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

    PubMed

    Salsbury, Freddie R; Knutson, Stacy T; Poole, Leslie B; Fetrow, Jacquelyn S

    2008-02-01

    Cysteine sulfenic acid (Cys-SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. This post-translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys-SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKaS of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys-SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites.

  17. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  18. Proteome-wide analysis of nonsynonymous single-nucleotide variations in active sites of human proteins.

    PubMed

    Dingerdissen, Hayley; Motwani, Mona; Karagiannis, Konstantinos; Simonyan, Vahan; Mazumder, Raja

    2013-03-01

    An enzyme's active site is essential to normal protein activity such that any disruptions at this site may lead to dysfunction and disease. Nonsynonymous single-nucleotide variations (nsSNVs), which alter the amino acid sequence, are one type of disruption that can alter the active site. When this occurs, it is assumed that enzyme activity will vary because of the criticality of the site to normal protein function. We integrate nsSNV data and active site annotations from curated resources to identify all active-site-impacting nsSNVs in the human genome and search for all pathways observed to be associated with this data set to assess the likely consequences. We find that there are 934 unique nsSNVs that occur at the active sites of 559 proteins. Analysis of the nsSNV data shows an over-representation of arginine and an under-representation of cysteine, phenylalanine and tyrosine when comparing the list of nsSNV-impacted active site residues with the list of all possible proteomic active site residues, implying a potential bias for or against variation of these residues at the active site. Clustering analysis shows an abundance of hydrolases and transferases. Pathway and functional analysis shows several pathways over- or under-represented in the data set, with the most significantly affected pathways involved in carbohydrate metabolism. We provide a table of 32 variation-substrate/product pairs that can be used in targeted metabolomics experiments to assay the effects of specific variations. In addition, we report the significant prevalence of aspartic acid to histidine variation in eight proteins associated with nine diseases including glycogen storage diseases, lacrimo-auriculo-dento-digital syndrome, Parkinson's disease and several cancers.

  19. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  20. Site and Orbit Repeatabilities using Adaptive Mapping Functions

    NASA Astrophysics Data System (ADS)

    Desjardins, Camille; Gegout, Pascal; Soudarin, Laurent; Biancale, Richard; Perosanz, Felix

    2015-04-01

    The electromagnetic signals emitted by the satellite positioning systems travel at the speed of light in a straight line in a vacuum but are modified in their propagation through the neutral atmosphere by temporal and spatial changes of density, and composition and refractivity. These waves are slowed down and their trajectories are bent. This presentation summarizes the performances of the modeling of the tropospheric propagation by the ray tracing technique through the assimilations of the European Meteorological Centre (ECMWF) in the framework of realizing the geodetic reference frame. This goal is achieved by modeling the spatial variability of the propagation using the time variable three-dimensional physical parameters of the atmosphere. The tropospheric delays obtained by ray tracing in all directions throughout the meteorological model surrounding the geodetic site, are fitted by Adaptive Mapping Functions (AMF) parameterized by several tens of coefficients. The delays produced by the Horizon software are then experimented, kept unchanged or adjusted, when recovering a reference frame based on hundred sites using the GINS software. Without any adjustments of the tropospheric modeling, the subcentimetric performances of the AMF are demonstrated by the repeatability of sites positions and GPS satellites orbits. When some AMF coefficients are adjusted, the accuracy of orbits recovery in term of quadratic mean is 7 to 8 millimeters. This limit is imposed by the lack or deficiency of other models, such as non-tidal and tidal loading respectively. Hence the repeatability of the vertical position is not enhanced by changing the propagation model. At the contrary, the repeatability of the horizontal position of geodetic sites is greatly enhanced by accounting for the azimuthal variability provided by the realistic 3D shapes of the Atmosphere and the Earth and the rigorous interpolations of atmospheric parameters included in Adaptive Mapping Functions with respect

  1. 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

    2016-09-06

    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.

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

    PubMed Central

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

    2016-01-01

    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

  3. A functional study of concanavalin A-histamine binding site overlap in Tetrahymena phagocytosis test.

    PubMed

    Csaba, G; Darvas, Z; László, V

    1983-01-01

    Treatment with histamine stimulated the phagocytotic activity of the Tetrahymena to a measurable degree, which was still demonstrable after a week (about 40 generations). Concanavalin A, which binds to the same membrane binding site as histamine, inhibited the stimulatory action of subsequently added histamine, but did not in itself influence phagocytotic activity in any way. The inhibitory effect of Con A on the histamine binding site proved to be dose-dependent. These observations stress the importance of investigating the functional context--as sole realistic measure--of receptor--ligand bindings.

  4. Functional classification of protein 3D structures from predicted local interaction sites.

    PubMed

    Parasuram, Ramya; Lee, Joslynn S; Yin, Pengcheng; Somarowthu, Srinivas; Ondrechen, Mary Jo

    2010-12-01

    A new approach to the functional classification of protein 3D structures is described with application to some examples from structural genomics. This approach is based on functional site prediction with THEMATICS and POOL. THEMATICS employs calculated electrostatic potentials of the query structure. POOL is a machine learning method that utilizes THEMATICS features and has been shown to predict accurate, precise, highly localized interaction sites. Extension to the functional classification of structural genomics proteins is now described. Predicted functionally important residues are structurally aligned with those of proteins with previously characterized biochemical functions. A 3D structure match at the predicted local functional site then serves as a more reliable predictor of biochemical function than an overall structure match. Annotation is confirmed for a structural genomics protein with the ribulose phosphate binding barrel (RPBB) fold. A putative glucoamylase from Bacteroides fragilis (PDB ID 3eu8) is shown to be in fact probably not a glucoamylase. Finally a structural genomics protein from Streptomyces coelicolor annotated as an enoyl-CoA hydratase (PDB ID 3g64) is shown to be misannotated. Its predicted active site does not match the well-characterized enoyl-CoA hydratases of similar structure but rather bears closer resemblance to those of a dehalogenase with similar fold.

  5. Functionalized active-nucleus complex sensor

    DOEpatents

    Pines, Alexander; Wemmer, David E.; Spence, Megan; Rubin, Seth

    2003-11-25

    A functionalized active-nucleus complex sensor that selectively associates with one or more target species, and a method for assaying and screening for one or a plurality of target species utilizing one or a plurality of functionalized active-nucleus complexes with at least two of the functionalized active-nucleus complexes having an attraction affinity to different corresponding target species. The functionalized active-nucleus complex has an active-nucleus and a targeting carrier. The method involves functionalizing an active-nucleus, for each functionalized active-nucleus complex, by incorporating the active-nucleus into a macromolucular or molecular complex that is capable of binding one of the target species and then bringing the macromolecular or molecular complexes into contact with the target species and detecting the occurrence of or change in a nuclear magnetic resonance signal from each of the active-nuclei in each of the functionalized active-nucleus complexes.

  6. 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.

  7. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism.

    PubMed

    Medina-Carmona, Encarnación; Neira, Jose L; Salido, Eduardo; Fuchs, Julian E; Palomino-Morales, Rogelio; Timson, David J; Pey, Angel L

    2017-03-14

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

  8. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

    PubMed Central

    Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

    2017-01-01

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention. PMID:28291250

  9. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

    NASA Astrophysics Data System (ADS)

    Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

    2017-03-01

    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

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

    PubMed Central

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

    2015-01-01

    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 (Mw 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

  11. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  12. Maintenance of plastid RNA editing activities independently of their target sites.

    PubMed

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-03-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell.

  13. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

    PubMed Central

    Breunig, K D; Kuger, P

    1987-01-01

    As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription. Images PMID:2830492

  14. Incorporating distant sequence features and radial basis function networks to identify ubiquitin conjugation sites.

    PubMed

    Lee, Tzong-Yi; Chen, Shu-An; Hung, Hsin-Yi; Ou, Yu-Yen

    2011-03-09

    Ubiquitin (Ub) is a small protein that consists of 76 amino acids about 8.5 kDa. In ubiquitin conjugation, the ubiquitin is majorly conjugated on the lysine residue of protein by Ub-ligating (E3) enzymes. Three major enzymes participate in ubiquitin conjugation. They are E1, E2 and E3 which are responsible for activating, conjugating and ligating ubiquitin, respectively. Ubiquitin conjugation in eukaryotes is an important mechanism of the proteasome-mediated degradation of a protein and regulating the activity of transcription factors. Motivated by the importance of ubiquitin conjugation in biological processes, this investigation develops a method, UbSite, which uses utilizes an efficient radial basis function (RBF) network to identify protein ubiquitin conjugation (ubiquitylation) sites. This work not only investigates the amino acid composition but also the structural characteristics, physicochemical properties, and evolutionary information of amino acids around ubiquitylation (Ub) sites. With reference to the pathway of ubiquitin conjugation, the substrate sites for E3 recognition, which are distant from ubiquitylation sites, are investigated. The measurement of F-score in a large window size (-20∼+20) revealed a statistically significant amino acid composition and position-specific scoring matrix (evolutionary information), which are mainly located distant from Ub sites. The distant information can be used effectively to differentiate Ub sites from non-Ub sites. As determined by five-fold cross-validation, the model that was trained using the combination of amino acid composition and evolutionary information performs best in identifying ubiquitin conjugation sites. The prediction sensitivity, specificity, and accuracy are 65.5%, 74.8%, and 74.5%, respectively. Although the amino acid sequences around the ubiquitin conjugation sites do not contain conserved motifs, the cross-validation result indicates that the integration of distant sequence features of Ub

  15. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  16. 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

  17. 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)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.

  18. In vivo suppressive function of myeloid-derived suppressor cells is limited to the inflammatory site

    PubMed Central

    Haverkamp, Jessica M.; Crist, Scott A.; Elzey, Bennett D.; Cimen, Cansu; Ratliff, Timothy L.

    2011-01-01

    Current thinking suggests that despite the heterogeneity of myeloid-derived suppressor cells (MDSC), all Gr-1+CD11b+ cells can become suppressive when exposed to inflammatory stimuli. In vitro evaluation shows MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSC function enhances T cell responses. However, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T cell responses in vivo has not been directly evaluated. We now demonstrate that during a tissue specific inflammatory response, MDSC inhibition of CD8 T cell proliferation and IFN-γ production is restricted to the inflammatory site. Using a prostate specific inflammatory model and a heterotopic prostate tumor model, we show that MDSC from inflammatory sites or from tumor tissue possess immediate capacity to inhibit T cell function, whereas those isolated from peripheral tissues (spleens and liver) are not suppressive without activation of iNOS by exposure to IFN-γ. These data show MDSC are important regulators of immune responses in the prostate during acute inflammation and the chronic inflammatory setting of tumor growth and that regulation of T cell function by MDSC during a localized inflammatory response is restricted in vivo to the site of an ongoing immune response. PMID:21287554

  19. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  20. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites.

    PubMed

    Dian, Cyril; Vitale, Sylvia; Leonard, Gordon A; Bahlawane, Christelle; Fauquant, Caroline; Leduc, Damien; Muller, Cécile; de Reuse, Hilde; Michaud-Soret, Isabelle; Terradot, Laurent

    2011-03-01

    Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS₄ site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

  1. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

    PubMed Central

    Chappell, Stephen A.; Edelman, Gerald M.; Mauro, Vincent P.

    2000-01-01

    This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements. PMID:10677496

  2. Prediction of biological functions on glycosylation site migrations in human influenza H1N1 viruses.

    PubMed

    Sun, Shisheng; Wang, Qinzhe; Zhao, Fei; Chen, Wentian; Li, Zheng

    2012-01-01

    Protein glycosylation alteration is typically employed by various viruses for escaping immune pressures from their hosts. Our previous work had shown that not only the increase of glycosylation sites (glycosites) numbers, but also glycosite migration might be involved in the evolution of human seasonal influenza H1N1 viruses. More importantly, glycosite migration was likely a more effectively alteration way for the host adaption of human influenza H1N1 viruses. In this study, we provided more bioinformatics and statistic evidences for further predicting the significant biological functions of glycosite migration in the host adaptation of human influenza H1N1 viruses, by employing homology modeling and in silico protein glycosylation of representative HA and NA proteins as well as amino acid variability analysis at antigenic sites of HA and NA. The results showed that glycosite migrations in human influenza viruses have at least five possible functions: to more effectively mask the antigenic sites, to more effectively protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. The information here can provide some constructive suggestions for the function research related to protein glycosylation of influenza viruses, although these predictions still need to be supported by experimental data.

  3. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  4. 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.

  5. Site-directed mutagenesis of IRX9, IRX9L and IRX14 proteins involved in xylan biosynthesis: glycosyltransferase activity is not required for IRX9 function in Arabidopsis.

    PubMed

    Ren, Yanfang; Hansen, Sara Fasmer; Ebert, Berit; Lau, Jane; Scheller, Henrik Vibe

    2014-01-01

    Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic

  6. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  7. Site-specific methylated reporter constructs for functional analysis of DNA methylation.

    PubMed

    Han, Weiguo; Shi, Miao; Spivack, Simon D

    2013-11-01

    Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.

  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. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

  10. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.« less

  11. 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.

  12. Regulation of BRCA1 Function by DNA Damage-Induced Site-Specific Phosphorylation

    DTIC Science & Technology

    2005-06-01

    Rad51p proteins, the lat - end-joining. BRCA-2 complexed with machinery. ter a member of the RAD-52 epi- RAD-51 is active in strand exchange BRCA- 1 has...AD Award Number: DAMD17-02- 1 -0584 TITLE: Regulation of BRCAl Function by DNA Damage-Induced Site- Specific Phosphorylation PRINCIPAL INVESTIGATOR...ApprovedREPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of Information Is estimated to average 1 hour per

  13. Macrophage Biochemistry, Activation and Function

    DTIC Science & Technology

    1981-01-01

    glucoeidase +8 . . Sulfatase c +8 Modified from Morahan, 1980. b(+)Exhibit@ activity; (-) lacks activity; (+) weak or marginal activity. ’References: (1...endoplasmic reticulum enzymes, sulfatase c and alkaline a-glucosidase. Dissociation of the lysosomal enzyme patterns from sulfatase c and alkaline r...1974; Beaufay et al., 1974). Peritoneal macrophages are deficient or contain inauf- • -𔃼 :’- 41 ficient quantities of the classical constituents to be

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

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  15. 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…

  16. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  17. 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

  18. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  19. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    PubMed

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  20. 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.

  1. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  2. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary Uroporphyrinogen Decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of the four acetate side chains on the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer with the active site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single chain protein (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function, and suggest that the dimeric structure of URO-D is required to achieve conformational stability and create a large active site cleft. PMID:19362562

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

    SciTech Connect

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

    2009-01-01

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

  4. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  5. Transcriptional activation by LR1 at the Eµ enhancer and switch region sites

    PubMed Central

    Hanakahi, L. A.; Maizels, Nancy

    2000-01-01

    LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Eµ immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Eµ core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Eµ core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions. PMID:10908319

  6. Identification of functional regions in the Rhodospirillum rubrum L-asparaginase by site-directed mutagenesis.

    PubMed

    Pokrovskaya, M V; Aleksandrova, S S; Pokrovsky, V S; Veselovsky, A V; Grishin, D V; Abakumova, O Yu; Podobed, O V; Mishin, A A; Zhdanov, D D; Sokolov, N N

    2015-03-01

    Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

  7. Distribution bias analysis of germline and somatic single-nucleotide variations that impact protein functional site and neighboring amino acids

    PubMed Central

    Pan, Yang; Yan, Cheng; Hu, Yu; Fan, Yu; Pan, Qing; Wan, Quan; Torcivia-Rodriguez, John; Mazumder, Raja

    2017-01-01

    Single nucleotide variations (SNVs) can result in loss or gain of protein functional sites. We analyzed the effects of SNVs on enzyme active sites, ligand binding sites, and various types of post translational modification (PTM) sites. We found that, for most types of protein functional sites, the SNV pattern differs between germline and somatic mutations as well as between synonymous and non-synonymous mutations. From a total of 51,138 protein functional site affecting SNVs (pfsSNVs), a pan-cancer analysis revealed 142 somatic pfsSNVs in five or more cancer types. By leveraging patient information for somatic pfsSNVs, we identified 17 loss of functional site SNVs and 60 gain of functional site SNVs which are significantly enriched in patients with specific cancer types. Of the key pfsSNVs identified in our analysis above, we highlight 132 key pfsSNVs within 17 genes that are found in well-established cancer associated gene lists. For illustrating how key pfsSNVs can be prioritized further, we provide a use case where we performed survival analysis showing that a loss of phosphorylation site pfsSNV at position 105 in MEF2A is significantly associated with decreased pancreatic cancer patient survival rate. These 132 pfsSNVs can be used in developing genetic testing pipelines. PMID:28176830

  8. Crystal structure of plant ferritin reveals a novel metal binding site that functions as a transit site for metal transfer in ferritin.

    PubMed

    Masuda, Taro; Goto, Fumiyuki; Yoshihara, Toshihiro; Mikami, Bunzo

    2010-02-05

    Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-A resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu(173) and carbonyl oxygen of Thr(168), which correspond, respectively, to Glu(140) and Thr(135) of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

  9. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  10. Multimorbidity, cognitive function, and physical activity.

    PubMed

    Loprinzi, Paul D

    2016-02-01

    Previous research demonstrates that both physical activity and multimorbidity are associated with cognitive function. However, the extent to which physical activity may moderate the relationship between multimorbidity and cognitive function has not been thoroughly evaluated. Data from the 1999-2002 NHANES were used (60+ years; N = 2157). A multimorbidity index variable was created based on physician diagnosis of a multitude of chronic diseases. Physical activity was self-reported and cognitive function was evaluated from the digit symbol substitution test. Multimorbidity was inversely associated with cognitive function for the unadjusted and adjusted models. However, generally, multimorbidity was no longer associated with cognitive function for the majority of older adults who achieved the minimum recommended physical activity level (≥2000 MET-min-month), as issued by the United States Department of Health and Human Services. In this national sample of older adults, there was some evidence to suggest that physical activity moderates the relationship between multimorbidity and cognitive function.

  11. Ground Truth and Application for the Anisotropic Receiver Functions Technique - Test site KTB: the installation campaign

    NASA Astrophysics Data System (ADS)

    Bianchi, Irene; Anselmi, Mario; Apoloner, Maria-Theresia; Qorbani, Ehsan; Gribovszki, Katalin; Bokelmann, Götz

    2015-04-01

    The project at hand is a field test around the KTB (Kontinentale Tiefbohrung) site in the Oberpfalz, Southeastern Germany, at the northwestern edge of the Bohemian Massif. The region has been extensively studied through the analysis of several seismic reflection lines deployed around the drilling site. The deep borehole had been placed into gneiss rocks of the Zone Erbendorf-Vohenstrauss. Drilling activity lasted since 1987 to 1994, and it descends down to a depth of 9101 meters. In our experiment, we aim to recover structural information as well as anisotropy of the upper crust using the receiver function technique. This retrieved information will form the base for a comparison between the resulting anisotropy amount and orientation with information of rock samples from up to 9 km depth, and with earlier high-frequency seismic experiments around the drill site. For that purpose, we installed 9 seismic stations, and recorded seismicity continuously for two years.

  12. Neuroprotection by a novel NMDAR functional glycine site partial agonist, GLYX-13.

    PubMed

    Stanton, Patric K; Potter, Pamela E; Aguilar, Jennifer; Decandia, Maria; Moskal, Joseph R

    2009-08-26

    GLYX-13 (threonine-proline-proline-threonine-amide) is an amidated di-pyrrolidine that acts as a functional partial agonist at the glycine site on N-methyl-D-aspartate glutamate receptors (NMDARs). GLYX-13 can both increase NMDAR conductance at NR2B-containing receptors, and reduce conductance of non-NR2B-containing receptors. Here, we report that GLYX-13 potently reduces delayed (24 h) death of CA1 pyramidal neurons produced by bilateral carotid occlusion in Mongolian gerbils, when administered up to 5 h post-ischemia. GLYX-13 also reduced delayed (24 h) neuronal death of CA1, CA3, and dentate gyrus principal neurons elicited by oxygen/glucose deprivation in in-vitro hippocampal organotypic slice cultures, when applied up to 2 h post-oxygen/glucose deprivation. The glycine site full agonist D-serine completely occluded neuroprotection, indicating that GLYX-13 acts by modulating activation of this site.

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

    SciTech Connect

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

    2009-01-01

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

  14. Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo

    SciTech Connect

    Girard, C.; Brodeur, J.C.; Hontela, A.

    1995-12-31

    The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

  15. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  16. 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.

  17. Di/mono-nuclear iron(I)/(II) complexes as functional models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases: protonations, molecular structures and electrochemical properties.

    PubMed

    Gao, Shang; Fan, Jiangli; Sun, Shiguo; Song, Fengling; Peng, Xiaojun; Duan, Qian; Jiang, Dayong; Liang, Qingcheng

    2012-10-21

    Di/mono-nuclear iron(I)/(II) complexes containing conjugated and electron-withdrawing S-to-S linkers, [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(6)] (1), [{(μ-S)(2)(C(4)N(2)H(2))}Fe(2)(CO)(5)(PMe(3))] (1P), and [{(μ-S)(2)(C(4)N(2)H(2))}Fe(CO)(2)(PMe(3))(2)] (2) were prepared as biomimetic models for the 2Fe2S subunit and distal Fe moiety of the active site of [FeFe] hydrogenases. The N atoms in the heterocyclic pyrazines of 1 and 2 were protonated in the presence of proton acid to generate one and two hydrides, [1(NH)](+) CF(3)SO(3)(-), [2(NH)](+) CF(3)SO(3)(-), and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2), respectively. The protonation processes were evidenced by in situ IR and NMR spectroscopy. The molecular structures of the protonated species [1(NH)](+) CF(3)SO(3)(-) and [2(NH)(2)](2+) (CF(3)SO(3)(-))(2) together with their originating complexes and , and the mono-PMe(3) substituted diiron complex were identified by X-ray crystallography. The IR and single-crystal analysis data all suggested that the electron-withdrawing bridge, pyrazine, led to decreased electron density at the Fe centers of the model complexes, which was consistent with the electrochemical studies. The cyclic voltammograms indicated that complex exhibited a low primary reduction potential at -1.17 V vs. Fc-Fc(+) with a 270 mV positive shift compared with that of the benzene-1,2-dithiolate (bdt) bridged analogue [(μ-bdt)Fe(2)(CO)(6)]. Under the weak acid conditions, complexes 1 and 2 could electrochemically catalyze the proton reduction. More interestingly, the mononuclear ferrous complex 2 showed two catalytic peaks during the formation of hydrogen, confirming its potential as a catalyst for hydrogen production.

  18. Dual function of a nuclear factor I binding site in MMTV transcription regulation.

    PubMed Central

    Buetti, E; Kühnel, B; Diggelmann, H

    1989-01-01

    Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. Images PMID:2542892

  19. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  20. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  1. 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.

  2. 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

  3. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  4. 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.

  5. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  6. Are Synonymous Sites in Primates and Rodents Functionally Constrained?

    PubMed

    Price, Nicholas; Graur, Dan

    2016-01-01

    It has been claimed that synonymous sites in mammals are under selective constraint. Furthermore, in many studies the selective constraint at such sites in primates was claimed to be more stringent than that in rodents. Given the larger effective population sizes in rodents than in primates, the theoretical expectation is that selection in rodents would be more effective than that in primates. To resolve this contradiction between expectations and observations, we used processed pseudogenes as a model for strict neutral evolution, and estimated selective constraint on synonymous sites using the rate of substitution at pseudosynonymous and pseudononsynonymous sites in pseudogenes as the neutral expectation. After controlling for the effects of GC content, our results were similar to those from previous studies, i.e., synonymous sites in primates exhibited evidence for higher selective constraint that those in rodents. Specifically, our results indicated that in primates up to 24% of synonymous sites could be under purifying selection, while in rodents synonymous sites evolved neutrally. To further control for shifts in GC content, we estimated selective constraint at fourfold degenerate sites using a maximum parsimony approach. This allowed us to estimate selective constraint using mutational patterns that cause a shift in GC content (GT ↔ TG, CT ↔ TC, GA ↔ AG, and CA ↔ AC) and ones that do not (AT ↔ TA and CG ↔ GC). Using this approach, we found that synonymous sites evolve neutrally in both primates and rodents. Apparent deviations from neutrality were caused by a higher rate of C → A and C → T mutations in pseudogenes. Such differences are most likely caused by the shift in GC content experienced by pseudogenes. We conclude that previous estimates according to which 20-40% of synonymous sites in primates were under selective constraint were most likely artifacts of the biased pattern of mutation.

  7. Control of the active site structure of giant bilayer hemoglobin from the Annelid Eisenia foetida using hierarchic assemblies

    SciTech Connect

    Girasole, Marco; Arcovito, Alessandro; Marconi, Augusta; Davoli, Camilla; Congiu-Castellano, Agostina; Bellelli, Andrea; Amiconi, Gino

    2005-12-05

    The active site structure of the oxygenated derivative of the main subassemblies (whole protein, dodecamers, and trimers) of the giant haemoglobin from Eisenia foetida has been characterized by x-ray absorption near edge structure spectroscopy. The data revealed a remarkable effect of the hierarchic assemblies on the active site of the subunit. Specifically, the whole protein has the same site structure of the dodecamer, while a sharp conformational transition occurs when the dodecamer is disassembled into trimers (and monomers) revealing that constraints due to the protein matrix determine the active site geometry and, consequently, the protein function in these large complexes.

  8. Geo-Chip analysis reveals reduced functional diversity of the bacterial community at a dumping site for dredged Elbe sediment.

    PubMed

    Störmer, Rebecca; Wichels, Antje; Gerdts, Gunnar

    2013-12-15

    The dumping of dredged sediments represents a major stressor for coastal ecosystems. The impact on the ecosystem function is determined by its complexity not easy to assess. In the present study, we evaluated the potential of bacterial community analyses to act as ecological indicators in environmental monitoring programmes. We investigated the functional structure of bacterial communities, applying functional gene arrays (GeoChip4.2). The relationship between functional genes and environmental factors was analysed using distance-based multivariate multiple regression. Apparently, both the function and structure of the bacterial communities are impacted by dumping activities. The bacterial community at the dumping centre displayed a significant reduction of its entire functional diversity compared with that found at a reference site. DDX compounds separated bacterial communities of the dumping site from those of un-impacted sites. Thus, bacterial community analyses show great potential as ecological indicators in environmental monitoring.

  9. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

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

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  11. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

  12. Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.

    PubMed Central

    Zeng, C; Pinsonneault, J; Gellon, G; McGinnis, N; McGinnis, W

    1994-01-01

    How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E. Images PMID:7910795

  13. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  14. 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.

  15. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  16. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  17. The Hanford Site multiple function badge demonstration project

    SciTech Connect

    Nelson, R.A.

    1993-03-01

    The US Department of Energy Hanford Site is an original defense production site dating from World War II. The US Department of Energy assigned a new waste management mission for the Hanford Site. This mission requires fewer manned security posts and more areas restricted because of safety and environmental concerns. Entry into these areas requires specific safety and environmental qualifications, which could be controlled by automated access control systems. Automated entry reduces costs of managing the Site but requires health physics, safety, employee training, security databases integration, and a network system. Another approach, avoiding integration, is individuals carrying this information on their person as a picture identity badge and access control token. The health physics, safety, employee training, and security regulations that define access requirements may be best served using a smart card as the access control token. This paper discusses the need for information distribution and automated access control, potential approaches for solving the need, and smart card work at the Hanford Site.

  18. The low-affinity Ca2(+)-binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction.

    PubMed Central

    Sweeney, H L; Brito, R M; Rosevear, P R; Putkey, J A

    1990-01-01

    Both troponin C (TnC) and calmodulin share a remarkably similar tertiary motif that may be common to other Ca2(+)-binding proteins with activator activity. TnC plays a critical role in regulating muscle contraction and is particularly well-suited for structural analysis by site-directed mutation. Fast-twitch skeletal muscle TnC has two low-affinity Ca2(+)-binding sites (sites I and II), while in cardiac and slow-twitch skeletal muscle TnC site I is inactive. Recently, using protein engineering, we directly demonstrated that binding of Ca2+ to the low-affinity site(s) initiates muscle contraction. In the present study, we use mutagenesis to determine whether either of the low-affinity sites in cardiac TnC can trigger contraction in slow-twitch skeletal muscle fibers. In one Ca2(+)-binding mutant, Ca2(+)-binding to the dormant low-affinity site I was restored (CBM+I). In a second mutant, site I was activated while site II was inactivated (CBM+I-IIA). Both proteins had the predicted CA2(+)-binding characteristics, and both were able to associate with troponin I and troponin T to form a troponin complex and integrate into permeabilized slow-twitch skeletal muscle fibers. A comparison of NMR spectra shows the aromatic regions in the two proteins to be qualitatively similar without divalent cations but markedly different with Ca2+. Mutant CBM+I supported force generation in skinned slow skeletal muscle fibers but had Sr2+ and Ca2+ sensitivities similar to fast skeletal TnC. Mutant CBM+I-IIA was unable to restore Ca2(+)-dependent contraction to TnC-depleted skinned slow muscle fibers. The data directly demonstrate that low-affinity sites I and II have distinct functions and that only site II in cardiac TnC can trigger muscle contraction in slow-twitch skeletal muscle fibers. This principle of distinct, modular activities for Ca2(+)-binding sites in the same protein may apply to other members of the TnC/calmodulin family. Images PMID:2263608

  19. Catalyst recognition of cis-1,2-diols enables site-selective functionalization of complex molecules

    NASA Astrophysics Data System (ADS)

    Sun, Xixi; Lee, Hyelee; Lee, Sunggi; Tan, Kian L.

    2013-09-01

    Carbohydrates and natural products serve essential roles in nature, and also provide core scaffolds for pharmaceutical agents and vaccines. However, the inherent complexity of these molecules imposes significant synthetic hurdles for their selective functionalization and derivatization. Nature has, in part, addressed these issues by employing enzymes that are able to orient and activate substrates within a chiral pocket, which increases dramatically both the rate and selectivity of organic transformations. In this article we show that similar proximity effects can be utilized in the context of synthetic catalysts to achieve general and predictable site-selective functionalization of complex molecules. Unlike enzymes, our catalysts apply a single reversible covalent bond to recognize and bind to specific functional group displays within substrates. By combining this unique binding selectivity and asymmetric catalysis, we are able to modify the less reactive axial positions within monosaccharides and natural products.

  20. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  1. 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

  2. Synthesis and application of functional branched macromolecules: From site isolation and energy harvesting to catalysis

    NASA Astrophysics Data System (ADS)

    Hecht, Stefan

    The symbiosis of our understanding of structure property relationships in many biological macromolecules and our increased ability to prepare large synthetic macromolecules with exquisite structural precision has generated a new area of research where chemistry and materials science join with biology. For example, numerous biological systems utilize the concept of site isolation whereby an active center or catalytic site is encapsulated, frequently within a protein, to afford properties that would not be encountered in the bulk state. The ability of a dendritic shell to encapsulate functional core moieties and to create specific site-isolated nanoenvironments, thereby affecting molecular properties, not only mimics natural systems but affords novel materials with unique characteristics. Furthermore, introduction of donor chromophores at periphery of dendrimers having a central acceptor dye enables spatial and spectral energy concentration at the core. Continuing the effort towards designing bio-inspired macromolecules, this dissertation describes the use of different polymer architectures to encapsulate active sites that have either photophysical, photochemical, or catalytic functions and the evaluation of site isolation using a variety of different techniques. While the first part is mainly concerned with different synthetic approaches towards site isolation of porphyrin moieties, the second part describes the design of light-driven catalytic systems incorporating both light harvesting and energy conversion. The fundamental knowledge that can be gleaned from such investigations has implications that range from the preliminary design of artificial enzymes to the construction of molecular-scale devices. After an overview of dendritically encapsulated functions (Chapter 1) and a brief account of a novel synthetic approach to benzene core dendrimers (Chapter 2), site isolation of porphyrin moieties within dendrimers, their linear structural isomers, and branched star

  3. An efficient perturbation method to predict the functionally key sites of glutamine binding protein.

    PubMed

    Lv, Dashuai; Wang, Cunxin; Li, Chunhua; Tan, Jianjun; Zhang, Xiaoyi

    2017-04-01

    Glutamine-Binding Protein (GlnBP) of Escherichia coli, an important member of the periplasmic binding protein family, is responsible for the first step in the active transport of glutamine across the cytoplasmic membrane. In this work, the functionally key regulation sites of GlnBP were identified by utilizing a perturbation method proposed by our group, in which the residues whose perturbations markedly change the binding free energy between GlnBP and glutamine are considered to be functionally key residues. The results show that besides the substrate binding sites, some other residues distant from the binding pocket, including the ones in the hinge regions between the two domains, the front- and back- door channels and the exposed region, are important for the function of glutamine binding and transport. The predicted results are well consistent with the theoretical and experimental data, which indicates that our method is an effective approach to identify the key residues important for both ligand binding and long-range allosteric signal transmission. This work can provide some insights into the function performance of GlnBP and the physical mechanism of its allosteric regulation.

  4. 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.

  5. 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.

  6. Computational approaches to find the active binding sites of biological targets against busulfan.

    PubMed

    Karthick, T; Tandon, Poonam

    2016-06-01

    Determination of electrophilic and nucleophilic sites of a molecule is the primary task to find the active sites of the lead molecule. In the present study, the active sites of busulfan have been predicted by molecular electrostatic potential surface and Fukui function analysis with the help of dispersion corrected density functional theory. Similarly, the identification of active binding sites of the proteins against lead compound plays a vital role in the field of drug discovery. Rigid and flexible molecular docking approaches are used for this purpose. For rigid docking, Hex 8.0.0 software employing fast Fourier transform (FFT) algorithm has been used. The partial flexible blind docking simulations have been performed with AutoDock 4.2 software; where a Lamarckian genetic algorithm is employed. The results showed that the most electrophilic atoms of busulfan bind with the targets. It is clear from the docking studies that busulfan has inhibition capability toward the targets 12CA and 1BZM. Graphical Abstract Docking of ligand and protein.

  7. C-H Activation on Co,O Sites: Isolated Surface Sites versus Molecular Analogs.

    PubMed

    Estes, Deven P; Siddiqi, Georges; Allouche, Florian; Kovtunov, Kirill V; Safonova, Olga V; Trigub, Alexander L; Koptyug, Igor V; Copéret, Christophe

    2016-11-16

    The activation and conversion of hydrocarbons is one of the most important challenges in chemistry. Transition-metal ions (V, Cr, Fe, Co, etc.) isolated on silica surfaces are known to catalyze such processes. The mechanisms of these processes are currently unknown but are thought to involve C-H activation as the rate-determining step. Here, we synthesize well-defined Co(II) ions on a silica surface using a metal siloxide precursor followed by thermal treatment under vacuum at 500 °C. We show that these isolated Co(II) sites are catalysts for a number of hydrocarbon conversion reactions, such as the dehydrogenation of propane, the hydrogenation of propene, and the trimerization of terminal alkynes. We then investigate the mechanisms of these processes using kinetics, kinetic isotope effects, isotopic labeling experiments, parahydrogen induced polarization (PHIP) NMR, and comparison with a molecular analog. The data are consistent with all of these reactions occurring by a common mechanism, involving heterolytic C-H or H-H activation via a 1,2 addition across a Co-O bond.

  8. Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils.

    PubMed

    Sprocati, Anna Rosa; Alisi, Chiara; Tasso, Flavia; Fiore, Alessia; Marconi, Paola; Langella, Francesca; Haferburg, Götz; Nicoara, Andrei; Neagoe, Aurora; Kothe, Erika

    2014-01-01

    The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon's index (alpha diversity) and Sørensen's Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

  9. 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

  10. 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.

  11. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  12. 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.

  13. 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.

  14. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

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

    2010-07-14

    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 A. 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.

  15. Identification of functional regions on the I-Ab molecule by site-directed mutagenesis.

    PubMed Central

    Cohn, L E; Glimcher, L H; Waldmann, R A; Smith, J A; Ben-Nun, A; Seidman, J G; Choi, E

    1986-01-01

    Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the beta 1 domain of the Ab beta gene we have constructed three structurally distinct mutant Ab beta genes. Each of these genes, as well as the wild-type Ab beta gene, was cotransfected together with the wild-type Ab alpha gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation of I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites. PMID:2418441

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

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    reaction center. Synchronously, Arg366 of the β sheet at the base holds the syn oxygen of the attacking carboxylic group so that the attack by the anti oxygen is feasible. This residue also contributes to the reduction of the unfavorable electrostatic potential at the reaction center. Present simulation clearly shows the catalytic role of the residues at reaction center. A precise and stable geometry of hydrogen bonded network develops within the active site, which is essential for the development of an optimum transition state geometry. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bond disappears. The serine binding site is most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn(2+) and Cys residues. Present simulation further suggests that the helix-turn-helix motif connecting the monomers might have important role in coordinating the functional dynamics of the two active sites. The N-terminal domain is involved in long-range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with tRNA. Overall conformational fluctuation is less in the N terminal compared to the catalytic domain due to the presence of a motif 2 loop, loop f, and serine ordering loop, which change conformation in the later domain during the reaction cycle. The dynamic perspective of the active site of (mk)SerRS with the mobile loop acting as the gate and dynamically silent β sheets performing as the base has similarity with the perception of the active site in various other enzymes.

  17. 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

  18. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  19. Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants.

    PubMed Central

    Roberts, G.; Berberian, G.; Beauge, L.

    1995-01-01

    We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (K0.5 between 500 and 1800 [mu]M). In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule. PMID:12228512

  20. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  1. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  2. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  3. Structure/Function/Dynamics of Photosystem II Plastoquinone Binding Sites

    PubMed Central

    Lambreva, Maya D.; Russo, Daniela; Polticelli, Fabio; Scognamiglio, Viviana; Antonacci, Amina; Zobnina, Veranika; Campi, Gaetano; Rea, Giuseppina

    2014-01-01

    Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as bio-sensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed. PMID:24678671

  4. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  5. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  6. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  7. 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.

  8. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  9. Mapping solvation dynamics at the function site of flavodoxin in three redox states.

    PubMed

    Chang, Chih-Wei; He, Ting-Fang; Guo, Lijun; Stevens, Jeffrey A; Li, Tanping; Wang, Lijuan; Zhong, Dongping

    2010-09-15

    Flavoproteins are unique redox coenzymes, and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant, and such an intimate relationship is essential to the biological function of interprotein electron transfer.

  10. 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.

  11. The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

    SciTech Connect

    Gopal, B.; Madan, Lalima L.; Betz, Stephen F.; Kossiakoff, Anthony A.

    2010-11-10

    Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.

  12. Opiates and cerebral functional activity in rats

    SciTech Connect

    Trusk, T.C.

    1986-01-01

    Cerebral activity was measured using the free-fatty acid (1-/sup 14/C) octanoate as a fast functional tracer in conscious, unrestrained rats 5 minutes after intravenous injection of heroin, cocaine or saline vehicle. Regional changes of octanoate labeling density in the autoradiograms relative to saline-injected animals were used to determine the functional activity effects of each drug. Heroin and cocaine each produced a distinctive pattern of activity increases and suppression throughout the rat brain. Similar regional changes induced by both drugs were found in limbic brain regions implicated in drug reinforcement. Labeled octanoate autoradiography was used to measure the cerebral functional response to a tone that had previously been paired to heroin injections. Rats were trained in groups of three consisting of one heroin self-administration animal, and two animals receiving yoked infusion of heroin or saline. A tone was paired with each infusion during training. Behavioral experiments in similarly trained rats demonstrated that these training conditions impart secondary reinforcing properties to the tone in animals previously self-administering heroin, while the tone remains behaviorally neutral in yoked-infusion rats. Cerebral functional activity was measured during presentation of the tone without drug infusion. Octanoate labeling density changed in fifteen brain areas in response to the tone previously paired to heroin without response contingency. Labeling density was significantly modified in sixteen regions as a result of previously pairing the tone to response-contingent heroin infusions.

  13. Cerebral blood flow response to functional activation

    PubMed Central

    Paulson, Olaf B; Hasselbalch, Steen G; Rostrup, Egill; Knudsen, Gitte Moos; Pelligrino, Dale

    2010-01-01

    Cerebral blood flow (CBF) and cerebral metabolic rate are normally coupled, that is an increase in metabolic demand will lead to an increase in flow. However, during functional activation, CBF and glucose metabolism remain coupled as they increase in proportion, whereas oxygen metabolism only increases to a minor degree—the so-called uncoupling of CBF and oxidative metabolism. Several studies have dealt with these issues, and theories have been forwarded regarding the underlying mechanisms. Some reports have speculated about the existence of a potentially deficient oxygen supply to the tissue most distant from the capillaries, whereas other studies point to a shift toward a higher degree of non-oxidative glucose consumption during activation. In this review, we argue that the key mechanism responsible for the regional CBF (rCBF) increase during functional activation is a tight coupling between rCBF and glucose metabolism. We assert that uncoupling of rCBF and oxidative metabolism is a consequence of a less pronounced increase in oxygen consumption. On the basis of earlier studies, we take into consideration the functional recruitment of capillaries and attempt to accommodate the cerebral tissue's increased demand for glucose supply during neural activation with recent evidence supporting a key function for astrocytes in rCBF regulation. PMID:19738630

  14. Functional activities of the amygdala: an overview.

    PubMed Central

    Rasia-Filho, A A; Londero, R G; Achaval, M

    2000-01-01

    Research to date into the amygdala shows that it has an integrative role in behavioural, vegetative and endocrine activities of animals in their relation with their environment. Animal studies show that amygdala has a role in emotional response, integrating input signals and initiating activities related to them. Different nuclei seem to have different effects. A complete picture of the functional roles of the amygdala is unavailable, and it has been suggested that the amygdala is functionally and anatomically heterogeneous. Amygdaloid subnuclei appear to have a role in the modulation of fear, in memory and attention, and in some sexual and sex-related behaviour of rats. In humans, functional magnetic resonance imaging shows that the amygdala responds preferentially to emotionally charged stimuli. Bilateral amygdala damage in humans can compromise the recognition of fear in facial expressions, an important ability in social judgement. Future study of the amygdala promises to shed light on emotional disorders in humans. PMID:10721680

  15. A split active site couples cap recognition by Dcp2 to activation

    PubMed Central

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site. PMID:20711189

  16. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  17. Toward a Functional Definition of Methane Super-Emitters: Application to Natural Gas Production Sites.

    PubMed

    Zavala-Araiza, Daniel; Lyon, David; Alvarez, Ramón A; Palacios, Virginia; Harriss, Robert; Lan, Xin; Talbot, Robert; Hamburg, Steven P

    2015-07-07

    Emissions from natural gas production sites are characterized by skewed distributions, where a small percentage of sites-commonly labeled super-emitters-account for a majority of emissions. A better characterization of super-emitters is needed to operationalize ways to identify them and reduce emissions. We designed a conceptual framework that functionally defines superemitting sites as those with the highest proportional loss rates (methane emitted relative to methane produced). Using this concept, we estimated total methane emissions from natural gas production sites in the Barnett Shale; functionally superemitting sites accounted for roughly three-fourths of total emissions. We discuss the potential to reduce emissions from these sites, under the assumption that sites with high proportional loss rates have excess emissions resulting from abnormal or otherwise avoidable operating conditions, such as malfunctioning equipment. Because the population of functionally superemitting sites is not expected to be static over time, continuous monitoring will likely be necessary to identify them and improve their operation. This work suggests that achieving and maintaining uniformly low emissions across the entire population of production sites will require mitigation steps at a large fraction of sites.

  18. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  19. Controlling activation site density by low-energy far-field stimulation in cardiac tissue.

    PubMed

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites ("virtual electrodes") in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  20. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  1. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

  2. Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase.

    PubMed

    Mercaldi, Gustavo F; Pereira, Humberto M; Cordeiro, Artur T; Michels, Paul A M; Thiemann, Otavio H

    2012-06-01

    Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

  3. Mechanism and Site Selectivity in Visible-Light Photocatalyzed C-H Functionalization: Insights from DFT Calculations.

    PubMed

    Demissie, Taye B; Hansen, Jørn H

    2016-08-19

    Visible-light photocatalyzed (VLPC) late-stage C-H functionalization is a powerful addition to the chemical synthesis toolkit. VLPC has a demonstrated potential for discovery of elusive and valuable transformations, particularly in functionalization of bioactive heterocycles. In order to fully harvest the potential of VLPC in the context of complex molecule synthesis, a thorough understanding of the elementary processes involved is crucial. This would enable more rational design of suitable reagents and catalysts, as well as prediction of activated C-H sites for functionalization. Such knowledge is essential when VLPC is to be employed in retrosynthetic analysis of complex molecules. Herein, we present a density functional theory (DFT) study of mechanistic details in the C-H functionalization of bioactive heterocycles exemplified by the methylation of the antifungal agent voriconazole. Moreover, we show that readily computed atomic charges can predict major site-selectivity in good agreement with experimental studies and thus be informative tools for the identification of active C-H functionalization sites in synthetic planning.

  4. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

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

    2010-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 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 ~5-fold and decreases in the rate constant for product release of ~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

  5. 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.

  6. 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.

  7. 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

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

    PubMed

    de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

    2015-12-01

    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 GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) 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 Ca(2+) 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 GABA(A) 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 Na(v)-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 GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

  9. CO-dynamics in the active site of cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Soloviov, Maksym; Meuwly, Markus

    2014-04-01

    The transfer of CO from heme a3 to the CuB site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the CuB site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ≈90% of the ligands are found to rebind to either the CuB (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet.

  10. Site-selective and stereoselective functionalization of unactivated C-H bonds

    NASA Astrophysics Data System (ADS)

    Liao, Kuangbiao; Negretti, Solymar; Musaev, Djamaladdin G.; Bacsa, John; Davies, Huw M. L.

    2016-05-01

    The laboratory synthesis of complex organic molecules relies heavily on the introduction and manipulation of functional groups, such as carbon-oxygen or carbon-halogen bonds; carbon-hydrogen bonds are far less reactive and harder to functionalize selectively. The idea of C-H functionalization, in which C-H bonds are modified at will instead of the functional groups, represents a paradigm shift in the standard logic of organic synthesis. For this approach to be generally useful, effective strategies for site-selective C-H functionalization need to be developed. The most practical solutions to the site-selectivity problem rely on either intramolecular reactions or the use of directing groups within the substrate. A challenging, but potentially more flexible approach, would be to use catalyst control to determine which site in a particular substrate would be functionalized. Here we describe the use of dirhodium catalysts to achieve highly site-selective, diastereoselective and enantioselective C-H functionalization of n-alkanes and terminally substituted n-alkyl compounds. The reactions proceed in high yield, and functional groups such as halides, silanes and esters are compatible with this chemistry. These studies demonstrate that high site selectivity is possible in C-H functionalization reactions without the need for a directing or anchoring group present in the molecule.

  11. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

    PubMed Central

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  12. Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors

    PubMed Central

    Estrada-Mondragon, Argel; Lynch, Joseph W.

    2015-01-01

    GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36′F sidechains to the “+” side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36′ methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites. PMID:26441518

  13. Deep Neural Networks with Multistate Activation Functions

    PubMed Central

    Cai, Chenghao; Xu, Yanyan; Ke, Dengfeng; Su, Kaile

    2015-01-01

    We propose multistate activation functions (MSAFs) for deep neural networks (DNNs). These MSAFs are new kinds of activation functions which are capable of representing more than two states, including the N-order MSAFs and the symmetrical MSAF. DNNs with these MSAFs can be trained via conventional Stochastic Gradient Descent (SGD) as well as mean-normalised SGD. We also discuss how these MSAFs perform when used to resolve classification problems. Experimental results on the TIMIT corpus reveal that, on speech recognition tasks, DNNs with MSAFs perform better than the conventional DNNs, getting a relative improvement of 5.60% on phoneme error rates. Further experiments also reveal that mean-normalised SGD facilitates the training processes of DNNs with MSAFs, especially when being with large training sets. The models can also be directly trained without pretraining when the training set is sufficiently large, which results in a considerable relative improvement of 5.82% on word error rates. PMID:26448739

  14. PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

    PubMed

    Urményi, T P; Van der Ploeg, L H

    1995-03-25

    In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

  15. Rna-seq analysis of the functional compartments within the rat placentation site

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rat placentation site is distinctly organized into interacting zones, the so-called labyrinth, junctional, and metrial gland compartments. These zones house unique cell populations equipped to undertake myriad prescribed functions including transport, hormonal responses, and immune interactions....

  16. Predicting functional divergence in protein evolution by site-specific rate shifts

    NASA Technical Reports Server (NTRS)

    Gaucher, Eric A.; Gu, Xun; Miyamoto, Michael M.; Benner, Steven A.

    2002-01-01

    Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the "non-homogeneous gamma model", which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

  17. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  18. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  19. Repair versus Checkpoint Functions of BRCA1 Are Differentially Regulated by Site of Chromatin Binding.

    PubMed

    Goldstein, Michael; Kastan, Michael B

    2015-07-01

    The product of the Brca1 tumor-suppressor gene is involved in multiple aspects of the cellular DNA damage response (DDR), including activation of cell-cycle arrests and DNA double-stranded break (DSB) repair by homologous recombination. Prior reports demonstrated that BRCA1 recruitment to areas of DNA breakage depended on RAP80 and the RNF8/RNF168 E3 ubiquitin ligases. Here, we extend these findings by showing that RAP80 is only required for the binding of BRCA1 to regions flanking the DSB, whereas BRCA1 binding directly to DNA breaks requires Nijmegen breakage syndrome 1 (NBS1). These differential recruitment mechanisms differentially affect BRCA1 functions: (i) RAP80-dependent recruitment of BRCA1 to chromatin flanking DNA breaks is required for BRCA1 phosphorylation at serine 1387 and 1423 by ATM and, consequently, for the activation of S and G(2) checkpoints; and (ii) BRCA1 interaction with NBS1 upon DSB induction results in an NBS1-dependent recruitment of BRCA1 directly to the DNA break and is required for nonhomologous end-joining repair. Together, these findings illustrate that spatially distinct fractions of BRCA1 exist at the DSB site, which are recruited by different mechanisms and execute different functions in the DDR.

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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.

  6. 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.

  7. Stochastic analysis of myoelectric temporal signatures for multifunctional single-site activation of prostheses and orthoses.

    PubMed

    Graupe, D; Salahi, J; Zhang, D S

    1985-01-01

    This paper is concerned with a stochastic time-series analysis of the temporal signatures of myoelectric (ME) signals including the determination of model order and sampling rate. The paper considers the use of time-series parameters for the activation of artificial limbs for high-level amputees, of stimulation electrodes or of powered braces for paralysed persons, in several degrees of freedom, from a single or two surface-electrode pairs at locations where considerable ME cross-talk exists. The multifunctional capability from a single site is based on the differences between the time-series (TS) parameters for different muscle activation patterns at the same ME site, these differences being thus used for limb function discrimination via easily trainable muscle activation patterns at the vicinity of the electrode site. Specifically, the analysis is in terms of identifying the AR parameters of a time-domain autoregressive (AR) signature model both for the complete ME spectrum and for parts thereof, and in terms of the autocorrelation of the signal and of the models residual. Determination of sampling rate and of model orders is discussed in detail. It is shown that, using online real-time analysis, differences in the AR time-series parameters can be observed for different trainable patterns of muscle activation, at the same electrode location, even at the same ME power levels, as long as considerable cross-talk exists at the electrode site. These parameter differences can be accentuated if one considers the AR parameters for lower-frequency spectral windows. A case is made in this paper for employing TS analysis to squeeze out information in a distinct but low-level ripple of the low frequency spectrum of the signal. This information tends to be ignored in frequency domain, but is all that the AR parameters care for in TS analysis, since they are not concerned, with a flat-average low-frequency spectrum, i.e., its white-noise-like part, which is the residual term

  8. Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2014-10-01

    Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

  9. 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

  10. Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides.

    PubMed

    Nitta, Y; Shirakawa, M; Takasaki, Y

    1996-01-01

    The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromi's theory, using (14)C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β -amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1,4 glucosidic linkage (kint) was five to seven times as large as those of the other enzymes.From the pH dependence of log[k0/Km], pK values of two functional ionizable groups were pK1 =4.0 and pK2 = 8.4. The pK values were 0.5-0.6 units for pK1 and 0.2-0.3 units for pK2 larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6kcal/mol larger than those of the other β-amylases.

  11. Lidar research activities and observations at NARL site, Gadanki, India

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

    The National Atmospheric Research Laboratory (NARL), a unit of Department of Space (DOS), located at Gadanki village (13.5°N, 79.2°E, 370 m AMSL) in India, is involved in the development of lidar remote sensing technologies for atmospheric research. Several advanced lidar technologies employing micropulse, polarization, Raman and scanning have been developed at this site and demonstrated for atmospheric studies during the period between 2008 and 2015. The technology of micropulse lidar, operates at 532 nm wavelength, was successfully transferred to an industry and the commercial version has been identified for Indian Lidar network (I-LINK) programme. Under this lidar network activity, several lidar units were installed at different locations in India to study tropospheric aerosols and clouds. The polarization sensitive lidar technology was realized using a set of mini photomultiplier tube (PMT) units and has the capability to operate during day and night without a pause. The lidar technology uses a compact flashlamp pumped Qswitched laser and employs biaxial configuration between the transmitter and receiver units. The lidar technology has been utilized for understanding the polarization characteristics of boundary layer aerosols during the mixed layer development. The demonstrated Raman lidar technology, uses the third harmonic wavelength of Nd:YAG laser, provides the altitude profiles of aerosol backscattering, extinction and water vapor covering the boundary layer range and allows operation during nocturnal periods. The Raman lidar derived height profiles of aerosol backscattering and extinction coefficient, lidar ratio, and watervapor mixing ratio inform the tropical boundary layer aerosol characteristics. The scanning lidar technology uses a near infrared laser wavelength for probing the lower atmosphere and has been utilized for high resolution cloud profiling during convective periods. The lidar technology is also used for rain rate measurement during

  12. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    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.

  13. Assessing the recovery of functional diversity after sustained sediment screening at an aggregate dredging site in the North Sea

    NASA Astrophysics Data System (ADS)

    Barrio Froján, Christopher R. S.; Cooper, Keith M.; Bremner, Julie; Defew, Emma C.; Wan Hussin, Wan M. R.; Paterson, David M.

    2011-05-01

    The effects of dredging the seabed for aggregate on benthic functional diversity were assessed using a suite of suitable indices on a recovering macrofaunal assemblage. Recovery was assessed as the return of a dredged assemblage to a state found in neighbouring undisturbed (reference) sites. In situ sediment screening was permitted during dredging operations; a difference in the sedimentary profile of the seabed between dredged and undisturbed reference sites was also observed. At sites of relatively high and low dredging intensity the sediment appeared more homogenous than reference sites after the selective removal of the coarser component. Initial assessment of the macrofaunal assemblage using univariate analytical techniques suggested a recovery of functional diversity at the low dredging intensity site after two years (according to the Infaunal Trophic Index, Taxonomic Distinctness index and Rao's Quadratic Entropy coefficient). However, multivariate analyses of the same data and of all indices except Taxonomic Distinctness indicated that assemblages at both high and low dredging intensity sites remained statistically indistinguishable from each other yet markedly different to the assemblage present in the reference area during the four-year study. The study concluded that recovery of functional diversity to a level found in a neighbouring undredged habitat had not occurred at either dredged site five years after the cessation of dredging. It is thought that the damage by dredging to functional diversity and to the capacity of the macrofaunal assemblage to recover is immediate and not so dependent on dredging intensity. The cumulative and wider ranging effects of sediment screening cannot be ignored or dismissed as a contributing factor to the similarities observed. The wider significance of these findings on the regulation of dredging activities is discussed.

  14. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  15. Pyrrolidine analogs of lobelane: Relationship of affinity for the dihydrotetrabenazine binding site with function of the vesicular monoamine transporter 2 (VMAT2)

    PubMed Central

    Vartak, Ashish P.; Nickell, Justin R.; Chagkutip, Jaturaporn; Dwoskin, Linda P.; Crooks, Peter A.

    2013-01-01

    Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogs that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure activity relationships indicate that structural modification within the pyrrolidine series resulted in analogs that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function. PMID:19691331

  16. Vestibular Function and Activities of Daily Living

    PubMed Central

    Harun, Aisha; Semenov, Yevgeniy R.; Agrawal, Yuri

    2015-01-01

    Objective: Vestibular dysfunction increases with age and is associated with mobility difficulties and fall risk in older individuals. We evaluated whether vestibular function influences the ability to perform activities of daily living (ADLs). Method: We analyzed the 1999 to 2004 National Health and Nutrition Examination Survey of adults aged older than 40 years (N = 5,017). Vestibular function was assessed with the Modified Romberg test. We evaluated the association between vestibular function and difficulty level in performing specific basic and instrumental ADLs, and total number of ADL impairments. Results: Vestibular dysfunction was associated with significantly higher odds of difficulty with nine ADLs, most strongly with difficulty managing finances (odds ratio [OR] = 2.64, 95% confidence interval [CI] = [1.18, 5.90]). In addition, vestibular dysfunction was associated with a significantly greater number of ADL impairments (β = .21, 95% CI = [0.09, 0.33]). This effect size was comparable with the influence of heavy smoking (β = .21, 95% CI = [0.06, 0.36]) and hypertension (β = .10, 95% CI = [0.02, 0.18]) on the number of ADL impairments. Conclusion: Vestibular dysfunction significantly influences ADL difficulty, most strongly with a cognitive rather than mobility-based task. These findings underscore the importance of vestibular inputs for both cognitive and physical daily activities. PMID:26753170

  17. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  18. Functions for diverse metabolic activities in heterochromatin

    PubMed Central

    Su, Xue Bessie; Pillus, Lorraine

    2016-01-01

    Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1’s functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes. PMID:26936955

  19. Functions for diverse metabolic activities in heterochromatin.

    PubMed

    Su, Xue Bessie; Pillus, Lorraine

    2016-03-15

    Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1's functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes.

  20. Remote site-selective C–H activation directed by a catalytic bifunctional template

    NASA Astrophysics Data System (ADS)

    Zhang, Zhipeng; Tanaka, Keita; Yu, Jin-Quan

    2017-03-01

    In chemical syntheses, the activation of carbon–hydrogen (C–H) bonds converts them directly into carbon–carbon or carbon–heteroatom bonds without requiring any prior functionalization. C–H activation can thus substantially reduce the number of steps involved in a synthesis. A single specific C–H bond in a substrate can be activated by using a ‘directing’ (usually a functional) group to obtain the desired product selectively. The applicability of such a C–H activation reaction can be severely curtailed by the distance of the C–H bond in question from the directing group, and by the shape of the substrate, but several approaches have been developed to overcome these limitations. In one such approach, an understanding of the distal and geometric relationships between the functional groups and C–H bonds of a substrate has been exploited to achieve meta-selective C–H activation by using a covalently attached, U-shaped template. However, stoichiometric installation of this template has not been feasible in the absence of an appropriate functional group on which to attach it. Here we report the design of a catalytic, bifunctional nitrile template that binds a heterocyclic substrate via a reversible coordination instead of a covalent linkage. The two metal centres coordinated to this template have different roles: one reversibly anchors substrates near the catalyst, and the other cleaves remote C–H bonds. Using this strategy, we demonstrate remote, site-selective C–H olefination of heterocyclic substrates that do not have the necessary functional groups for covalently attaching templates.

  1. 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.

  2. Effect of particle surface area on ice active site densities retrieved from droplet freezing spectra

    NASA Astrophysics Data System (ADS)

    Beydoun, Hassan; Polen, Michael; Sullivan, Ryan C.

    2016-10-01

    Heterogeneous ice nucleation remains one of the outstanding problems in cloud physics and atmospheric science. Experimental challenges in properly simulating particle-induced freezing processes under atmospherically relevant conditions have largely contributed to the absence of a well-established parameterization of immersion freezing properties. Here, we formulate an ice active, surface-site-based stochastic model of heterogeneous freezing with the unique feature of invoking a continuum assumption on the ice nucleating activity (contact angle) of an aerosol particle's surface that requires no assumptions about the size or number of active sites. The result is a particle-specific property g that defines a distribution of local ice nucleation rates. Upon integration, this yields a full freezing probability function for an ice nucleating particle. Current cold plate droplet freezing measurements provide a valuable and inexpensive resource for studying the freezing properties of many atmospheric aerosol systems. We apply our g framework to explain the observed dependence of the freezing temperature of droplets in a cold plate on the concentration of the particle species investigated. Normalizing to the total particle mass or surface area present to derive the commonly used ice nuclei active surface (INAS) density (ns) often cannot account for the effects of particle concentration, yet concentration is typically varied to span a wider measurable freezing temperature range. A method based on determining what is denoted an ice nucleating species' specific critical surface area is presented and explains the concentration dependence as a result of increasing the variability in ice nucleating active sites between droplets. By applying this method to experimental droplet freezing data from four different systems, we demonstrate its ability to interpret immersion freezing temperature spectra of droplets containing variable particle concentrations. It is shown that general

  3. Method and system to reclaim functional sites on a sorbent contaminated by heat stable salts

    DOEpatents

    Krutka, Holly; Sjostrom, Sharon; Morris, William J.

    2016-03-08

    The objective of this invention is to develop a method to reclaim functional sites on a CO.sub.2 sorbent that have reacted with an acid gas (other than CO.sub.2) to form heat stable salts (HSS). HSS are a significant concern for dry sorbent based CO.sub.2 capture because over time the buildup of HSS will reduce the overall functionality of the CO.sub.2 sorbent. A chemical treatment can remove the non-CO.sub.2 acid gas and reclaim functional sites that can then be used for further CO.sub.2 adsorption.

  4. Structure and function of ER membrane contact sites with other organelles.

    PubMed

    Phillips, Melissa J; Voeltz, Gia K

    2016-02-01

    The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

  5. Structure and function of ER membrane contact sites with other organelles

    PubMed Central

    Phillips, Melissa J.; Voeltz, Gia K.

    2016-01-01

    The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes. PMID:26627931

  6. 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.

  7. Exercise Sandals Increase Lower Extremity Electromyographic Activity During Functional Activities

    PubMed Central

    Hirth, Christopher J.; Guskiewicz, Kevin M.

    2003-01-01

    Objective: Anecdotal evidence suggests that use of Exercise Sandals results in a number of positive clinical outcomes. However, little research has been conducted to determine their efficacy objectively. Our purposes were to determine the effect of Exercise Sandals on lower leg electromyography (EMG) during activities in the Exercise Sandals and to compare EMG associated with Exercise Sandals with traditional lower extremity rehabilitation exercises. Design and Setting: Two within-subjects, repeated-measures designs were used to identify differences in lower extremity EMG: (1) between activities with and without Exercise Sandals and (2) between Exercise Sandals activities and traditional rehabilitation activities. All data were collected in the Sports Medicine Research Laboratory. Subjects: Eighteen subjects involved in rehabilitation using Exercise Sandals for at least 2 weeks within the year before data collection. Measurements: Mean EMG amplitudes from the tibialis anterior, peroneus longus, soleus, and lateral gastrocnemius muscles were measured during single-leg stance, side stepping, and “high knees,” all performed with and without the Exercise Sandals, as well as single-leg stance on a foam surface and T-band kicks in the sagittal and frontal planes. Results: Exercise Sandals increased lower leg EMG activity, particularly in the ankle invertors and evertors. Also, activities involving the Exercise Sandals resulted in EMG activity similar to or exceeding that associated with traditional ankle-rehabilitation exercises. Conclusions: These results, coupled with the fact that Exercise Sandals are used in a functional closed kinetic chain manner, suggest that they are an effective means of increasing lower extremity muscle activity. PMID:14608427

  8. Prediction of P53 Mutants (Multiple Sites) Transcriptional Activity Based on Structural (2D&3D) Properties

    PubMed Central

    Geetha Ramani, R.; Jacob, Shomona Gracia

    2013-01-01

    Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site) p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis. PMID:23468845

  9. Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

    PubMed Central

    Sun, Zhe; Martinez-Lemus, Luis A.; Hill, Michael A.; Meininger, Gerald A.

    2008-01-01

    Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2∼5 μm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of α5- and β3-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800∼1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to α5β1- and αvβ3-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces. PMID:18495809

  10. Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

    PubMed Central

    Sigala, Paul A.; Kraut, Daniel A.; Caaveiro, Jose M. M.; Pybus, Brandon; Ruben, Eliza A.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel

    2009-01-01

    Enzymes are classically proposed to accelerate reactions by binding substrates within active site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1 – 1 Å scale of most substrate rearrangements. The flexibility of active site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together high resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition state analog binding measurements to test the distance scale on which non-covalent forces can constrain side chain and ligand relaxation or translation along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function. PMID:18808119

  11. CO/Pt(111) : GGA density functional study of site preference for adsorption

    NASA Astrophysics Data System (ADS)

    Alaei, M.; Akbarzadeh, H.; Gholizadeh, H.; de Gironcoli, S.

    2008-02-01

    Based on density functional formalism, we investigate the site preference for the adsorption of CO on Cu, Rh, Ag, Pt, and Au(111) surfaces. The exchange-correlation term was approximated by BLYP, a functional within the generalized gradient approximation (GGA) family that combines Becke’s exchange functional with the Lee-Yang-Parr correlation functional. Our study shows that BLYP, although not a hybrid functional, can correctly predict the adsorption site for CO. This invalidate the general belief that only hybrid functionals are able to predict the correct site preference. We analyze our results by repeating all calculations using another well-known GGA functional, Perdew-Burke-Ernzerhof, which could trace back the origin of the success of BLYP to the different behavior of the two GGA functionals at large values of the reduced density gradients, s∝(∣∇n∣)/(n3/4) . This is a region where different functionals in the GGA family may legitimately behave very differently as it is poorly constrained on physical ground. Our present observation points to the possibility of further improving the accuracy of exchange-correlation functionals in the GGA family by properly constraining the remaining flexibility in the exchange-correlation kernel in this underdetermined region so as to reproduce known results in simple but critically important systems like the one studied here.

  12. Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues.

    PubMed

    Crabb, John W; O'Neil, June; Miyagi, Masaru; West, Karen; Hoff, Henry F

    2002-04-01

    Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 microM HNE lost approximately 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

  13. 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.

  14. Cluster Analysis of p53 Binding Site Sequences Reveals Subsets with Different Functions

    PubMed Central

    Lim, Ji-Hyun; Latysheva, Natasha S.; Iggo, Richard D.; Barker, Daniel

    2016-01-01

    p53 is an important regulator of cell cycle arrest, senescence, apoptosis and metabolism, and is frequently mutated in tumors. It functions as a tetramer, where each component dimer binds to a decameric DNA region known as a response element. We identify p53 binding site subtypes and examine the functional and evolutionary properties of these subtypes. We start with over 1700 known binding sites and, with no prior labeling, identify two sets of response elements by unsupervised clustering. When combined, they give rise to three types of p53 binding sites. We find that probabilistic and alignment-based assessments of cross-species conservation show no strong evidence of differential conservation between types of binding sites. In contrast, functional analysis of the genes most proximal to the binding sites provides strong bioinformatic evidence of functional differentiation between the three types of binding sites. Our results are consistent with recent structural data identifying two conformations of the L1 loop in the DNA binding domain, suggesting that they reflect biologically meaningful groups imposed by the p53 protein structure. PMID:27812278

  15. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

    PubMed Central

    Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

    2015-01-01

    Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

  16. An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

    PubMed

    Szewczak, A A; Ortoleva-Donnelly, L; Zivarts, M V; Oyelere, A K; Kazantsev, A V; Strobel, S A

    1999-09-28

    Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

  17. Functional diversity, soil features and community functioning: a test in a cerrado site.

    PubMed

    Freitas, J R; Cianciaruso, M V; Batalha, M A

    2012-08-01

    Community functioning may be affected by functional diversity, which measures the extent of complementarity in resource use. We tested whether there was a relationship between functional diversity of woody species and community functioning on a fine scale, using FD as a measure of functional diversity and litter decomposition rate as a surrogate for community functioning. We measured eight functional traits from a woodland cerrado community in southeastern Brazil. Then, we tested the correlation between FD and the decomposition rate taking into account differences in soil features and between decomposition rate and each trait separately. The decomposition rate was related to the aluminium and phosphorus concentration in soil, but not to FD, pointing out that functional diversity was not a good predictor of community functioning. There was a non-significant relationship between FD and the decomposition rate even when we considered each trait separately. Most studies in the relationships between biodiversity and community functioning on fine scales were carried out by experimental manipulation of diversity and in temperate regions. We carried out this fine scale study as a mensurative experiment and in a tropical savanna. Our findings indicated that the relationship between biodiversity and community functioning is not as straightforward as usually assumed.

  18. Structural and functional maturation of active zones in large synapses.

    PubMed

    Cano, Raquel; Torres-Benito, Laura; Tejero, Rocío; Biea, Anca I; Ruiz, Rocío; Betz, William J; Tabares, Lucía

    2013-02-01

    Virtually all functions of the nervous system rely upon synapses, the sites of communication between neurons and between neurons and other cells. Synapses are complex structures, each one comprising hundreds of different types of molecules working in concert. They are organized by adhesive and scaffolding molecules that align presynaptic vesicular release sites, namely, active zones, with postsynaptic neurotransmitter receptors, thereby allowing rapid and reliable intercellular communication. Most synapses are relatively small, and acting alone exerts little effect on their postsynaptic partners. Some, however, are much larger and stronger, reliably driving the postsynaptic cell to its action potential threshold, acting essentially as electrical relays of excitation. These large synapses are among the best understood, and two of these are the subject of this review, namely, the vertebrate neuromuscular junction and the calyx of Held synapse in the mammalian auditory pathway of the brain stem. Both synapses undergo through a complex and well-coordinated maturation process, during which time the molecular elements and the biophysical properties of the secretory machinery are continuously adjusted to the synapse size and to the functional requirements. We here review the morphological and functional changes occurring during postnatal maturation, noting particular similarities and differences between these two large synapses.

  19. Active membrane having uniform physico-chemically functionalized ion channels

    DOEpatents

    Gerald, II, Rex E; Ruscic, Katarina J; Sears, Devin N; Smith, Luis J; Klingler, Robert J; Rathke, Jerome W

    2012-09-24

    The present invention relates to a physicochemically-active porous membrane for electrochemical cells that purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. One dimension of the pore surface has a macroscopic length (1 nm-1000 .mu.m) and is directed parallel to the direction of an electric field, which is produced between the cathode and the anode electrodes of an electrochemical cell. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

  20. Innate response activator B cells: origins and functions

    PubMed Central

    Swirski, Filip K.

    2015-01-01

    Innate response activator (IRA) B cells are a subset of B-1a derived B cells that produce the growth factors granulocyte macrophage colony stimulating factor and IL-3. In mouse models of sepsis and pneumonia, B-1a B cells residing in serosal sites recognize bacteria, migrate to the spleen or lung, and differentiate to IRA B cells that then contribute to the host response by amplifying inflammation and producing polyreactive IgM. In atherosclerosis, IRA B cells accumulate in the spleen, where they promote extramedullary hematopoiesis and activate classical dendritic cells. In this review, we focus on the ontogeny and function of IRA B cells in acute and chronic inflammation. PMID:25957266

  1. 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)

  2. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  3. 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.

  4. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  5. Revealing the nature of the active site on the carbon catalyst for C-H bond activation.

    PubMed

    Sun, XiaoYing; Li, Bo; Su, Dangsheng

    2014-09-28

    A reactivity descriptor for the C-H bond activation on the nanostructured carbon catalyst is proposed. Furthermore the calculations reveal that the single ketone group can be an active site in ODH reaction.

  6. Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

    PubMed Central

    Exell, Jack C.; Thompson, Mark J.; Finger, L. David; Shaw, Steven J.; Debreczeni, Judit; Ward, Thomas A.; McWhirter, Claire; Siöberg, Catrine L. B.; Martinez Molina, Daniel; Mark Abbott, W.; Jones, Clifford D.; Nissink, J. Willem M.; Durant, Stephen T.; Grasby, Jane A.

    2016-01-01

    The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed. PMID:27526030

  7. 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.

  8. Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.

    PubMed

    Bourbon, M; Duarte, M A; Alves, A C; Medeiros, A M; Marques, L; Soutar, A K

    2009-05-01

    Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.

  9. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  10. MBSTAR: multiple instance learning for predicting specific functional binding sites in microRNA targets

    NASA Astrophysics Data System (ADS)

    Bandyopadhyay, Sanghamitra; Ghosh, Dip; Mitra, Ramkrishna; Zhao, Zhongming

    2015-01-01

    MicroRNA (miRNA) regulates gene expression by binding to specific sites in the 3'untranslated regions of its target genes. Machine learning based miRNA target prediction algorithms first extract a set of features from potential binding sites (PBSs) in the mRNA and then train a classifier to distinguish targets from non-targets. However, they do not consider whether the PBSs are functional or not, and consequently result in high false positive rates. This substantially affects the follow up functional validation by experiments. We present a novel machine learning based approach, MBSTAR (Multiple instance learning of Binding Sites of miRNA TARgets), for accurate prediction of true or functional miRNA binding sites. Multiple instance learning framework is adopted to handle the lack of information about the actual binding sites in the target mRNAs. Biologically validated 9531 interacting and 973 non-interacting miRNA-mRNA pairs are identified from Tarbase 6.0 and confirmed with PAR-CLIP dataset. It is found that MBSTAR achieves the highest number of binding sites overlapping with PAR-CLIP with maximum F-Score of 0.337. Compared to the other methods, MBSTAR also predicts target mRNAs with highest accuracy. The tool and genome wide predictions are available at http://www.isical.ac.in/~bioinfo_miu/MBStar30.htm.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  12. The installation campaign of 9 seismic stations around the KTB site to test anisotropy detection by the Receiver Function Technique

    NASA Astrophysics Data System (ADS)

    Bianchi, I.; Anselmi, M.; Apoloner, M. T.; Qorbani, E.; Gribovski, K.; Bokelmann, G.

    2015-09-01

    The project at hand is a field test around the KTB (Kontinentale Tiefbohrung) site in the Oberpfalz, Southeastern Germany, at the northwestern edge of the Bohemian Massif. The region has been extensively studied through the analysis of several seismic reflection lines deployed around the drilling site. The deep borehole had been placed into gneiss rocks of the Zone Erbendorf-Vohenstrauss. Drilling activity lasted from 1987 to 1994, and it descended down to a depth of 9101 m. In our experiment, we aim to recover structural information as well as anisotropy of the upper crust using the receiver function technique. This retrieved information is the basis for comparing the out-coming anisotropy amount and orientation with information of rock samples from up to 9 km depth, and with high-frequency seismic experiments around the drill site. For that purpose, we installed 9 seismic stations, and recorded seismicity continuously for two years from June 2012 to July 2014.

  13. Improving ATLAS grid site reliability with functional tests using HammerCloud

    NASA Astrophysics Data System (ADS)

    Elmsheuser, Johannes; Legger, Federica; Medrano Llamas, Ramon; Sciacca, Gianfranco; van der Ster, Dan

    2012-12-01

    With the exponential growth of LHC (Large Hadron Collider) data in 2011, and more coming in 2012, distributed computing has become the established way to analyse collider data. The ATLAS grid infrastructure includes almost 100 sites worldwide, ranging from large national computing centers to smaller university clusters. These facilities are used for data reconstruction and simulation, which are centrally managed by the ATLAS production system, and for distributed user analysis. To ensure the smooth operation of such a complex system, regular tests of all sites are necessary to validate the site capability of successfully executing user and production jobs. We report on the development, optimization and results of an automated functional testing suite using the HammerCloud framework. Functional tests are short lightweight applications covering typical user analysis and production schemes, which are periodically submitted to all ATLAS grid sites. Results from those tests are collected and used to evaluate site performances. Sites that fail or are unable to run the tests are automatically excluded from the PanDA brokerage system, therefore avoiding user or production jobs to be sent to problematic sites.

  14. Cardiovascular function following reduced aerobic activity

    NASA Technical Reports Server (NTRS)

    Raven, P. B.; Welch-O'Connor, R. M.; Shi, X.; Blomqvist, C. G. (Principal Investigator)

    1998-01-01

    PURPOSE: The aim of this study was to test the hypothesis that a sustained reduction of physical activity (deconditioning) would alter the cardiovascular regulatory function. METHODS: Nineteen young, healthy volunteers participated in physical deconditioning for a period of 8 wk. Before (pre) and following (post) physical deconditioning, the responses of heart rate (HR), mean arterial pressure (MAP, measured by Finapres), central venous pressure (CVP), stroke volume (SV, Doppler), and forearm blood flow (FBF, plethysmography) were determined during lower body negative pressure (LBNP). The carotid baroreflex (CBR) function was assessed using a train of pulsatile neck pressure (NP) and suction, and the aortic baroreflex control of HR was assessed during steady-state phenylephrine (PE) infusion superimposed by LBNP and NP to counteract the PE increased CVP and carotid sinus pressure, respectively. RESULTS: Active physical deconditioning significantly decreased maximal oxygen uptake (-7%) and LBNP tolerance (-13%) without a change in baseline hemodynamics. Plasma volume (-3% at P = 0.135), determined by Evans Blue dilution, and blood volume (-4% at P = 0.107) were not significantly altered. During LBNP -20 to -50 torr, there was a significantly greater drop of SV per unit decrease in CVP in the post- (14.7 +/- 1.6%/mm Hg) than predeconditioning (11.2 +/- 0.7%/mm Hg) test accompanied by a greater tachycardia. Deconditioning increased the aortic baroreflex sensitivity (pre vs post: -0.61 +/- 0.12 vs -0.84 +/- 0.14 bpm.mm-1 Hg, P = 0.009) and the slope of forearm vascular resistance (calculated from [MAP-CVP]/FBF) to CVP (-2.75 +/- 0.26 vs -4.94 +/- 0.97 PRU/mm Hg, P = 0.086). However, neither the CBR-HR (-0.28 +/- 0.03 VS -0.39 +/- 0.10 bpm.mm-1 Hg) nor the CBR-MAP (-0.37 +/- 0.16 vs -0.25 +/- 0.07 mm Hg/mm Hg) gains were statistically different between pre- and postdeconditioning. CONCLUSIONS: We concluded that the functional modification of the cardiac pressure

  15. A phosphorylation site in the ftz homeodomain is required for activity.

    PubMed Central

    Dong, J; Hung, L H; Strome, R; Krause, H M

    1998-01-01

    The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein. PMID:9545243

  16. Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis.

    PubMed

    Bolten, Marcel; Vahlensieck, Christian; Lipp, Colette; Leibundgut, Marc; Ban, Nenad; Weber-Ban, Eilika

    2017-03-10

    Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

  17. 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.

  18. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

    PubMed Central

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-01-01

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  19. 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.

  20. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine.

    PubMed

    Stec, Boguslaw

    2012-11-13

    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 CO(2). We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O(2) and CO(2) bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO(2) defines an elusive, preactivation complex that contains a metal cation Mg(2+) surrounded by three H(2)O/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.

  1. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  2. Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.

    PubMed

    Kakade, Poonam S; Budnar, Srikanth; Kalraiya, Rajiv D; Vaidya, Milind M

    2016-06-03

    Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

  3. Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

    PubMed Central

    Águila, Sonia; Teruel-Montoya, Raúl; Vicente, Vicente; Corral, Javier; Martínez-Martínez, Irene

    2016-01-01

    Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin. PMID:27322195

  4. Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites

    DTIC Science & Technology

    2013-08-01

    Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites Edward W. Malin, MD, Chaya M. Galin, BSN, RN... microcurrent in comparison to silver-coated dressing with sham microcurrent on wound-closure time for autogenous skin donor sites. Methods: Four...hundred five patients were screened for treatment of their donor sites using a silver-coated nylon dressing with either sham or active microcurrent

  5. 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

  6. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  7. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... from eligible active uranium and thorium processing site licensees for reimbursement under Title X of...). Title X requires DOE to reimburse eligible uranium and thorium licensees for certain costs...

  8. A model of the rabies virus glycoprotein active site.

    PubMed

    Rustici, M; Bracci, L; Lozzi, L; Neri, P; Santucci, A; Soldani, P; Spreafico, A; Niccolai, N

    1993-06-01

    The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

  9. Quantitative evaluation of activation state in functional brain imaging.

    PubMed

    Hu, Zhenghui; Ni, Pengyu; Liu, Cong; Zhao, Xiaohu; Liu, Huafeng; Shi, Pengcheng

    2012-10-01

    Neuronal activity can evoke the hemodynamic change that gives rise to the observed functional magnetic resonance imaging (fMRI) signal. These increases are also regulated by the resting blood volume fraction (V (0)) associated with regional vasculature. The activation locus detected by means of the change in the blood-oxygen-level-dependent (BOLD) signal intensity thereby may deviate from the actual active site due to varied vascular density in the cortex. Furthermore, conventional detection techniques evaluate the statistical significance of the hemodynamic observations. In this sense, the significance level relies not only upon the intensity of the BOLD signal change, but also upon the spatially inhomogeneous fMRI noise distribution that complicates the expression of the results. In this paper, we propose a quantitative strategy for the calibration of activation states to address these challenging problems. The quantitative assessment is based on the estimated neuronal efficacy parameter [Formula: see text] of the hemodynamic model in a voxel-by-voxel way. It is partly immune to the inhomogeneous fMRI noise by virtue of the strength of the optimization strategy. Moreover, it is easy to incorporate regional vascular information into the activation detection procedure. By combining MR angiography images, this approach can remove large vessel contamination in fMRI signals, and provide more accurate functional localization than classical statistical techniques for clinical applications. It is also helpful to investigate the nonlinear nature of the coupling between synaptic activity and the evoked BOLD response. The proposed method might be considered as a potentially useful complement to existing statistical approaches.

  10. Network analysis and protein function prediction with the PRODISTIN Web site.

    PubMed

    Baudot, Anaïs; Souiai, Ouissem; Brun, Christine

    2012-01-01

    Interactions between macromolecules are deciphered to gain information about biological processes and protein function. This information is hidden in large interaction networks, yet very complicated to dissect. In this context, the PRODISTIN Web site is dedicated to the clustering of network proteins according to the identity of their interaction partners, and to the subsequent functional annotation of these clusters. It allows analysing functionally networks and eventually leads to the prediction of function for uncharacterized protein based on their belonging to protein clusters. PRODISTIN analyses also provide an overview of the different biological processes existing in a given interactome. Here, we present a step-by-step procedure to analyse interaction networks using the PRODISTIN Web site. The protocol is illustrated by an application to the Campylobacter jejuni interactome.

  11. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

  12. Stone tool function at the paleolithic sites of Starosele and Buran Kaya III, Crimea: behavioral implications.

    PubMed

    Hardy, B L; Kay, M; Marks, A E; Monigal, K

    2001-09-11

    Stone tools are often the most abundant type of cultural remains at Paleolithic sites, yet their function is often poorly understood. Investigations of stone tool function, including microscopic use-wear and residue analyses, were performed on a sample of artifacts from the Paleolithic sites of Starosele (40,000-80,000 years BP) and Buran Kaya III (32,000-37,000 years BP). The Middle Paleolithic levels at Starosele exhibit a typical variant of the local Micoquian Industry. The artifacts from Buran Kaya III most closely resemble an Early Streletskayan Industry associated with the early Upper Paleolithic. The results of the functional analyses suggest that hominids at both sites were exploiting woody and starchy plant material as well as birds and mammals. Both sites show evidence of hafting of a wide variety of tools and the possible use of projectile or thrusting spears. These analyses were performed by using two different techniques conducted by independent researchers. Combined residue and use-wear analyses suggest that both the Upper Paleolithic and Middle Paleolithic hominids at these sites were broad-based foragers capable of exploiting a wide range of resources.

  13. Site Transfer Functions of Three-Component Ground Motion in Western Turkey

    NASA Astrophysics Data System (ADS)

    Ozgur Kurtulmus, Tevfik; Akyol, Nihal; Camyildiz, Murat; Gungor, Talip

    2015-04-01

    Because of high seismicity accommodating crustal deformation and deep graben structures, on which have, urbanized and industrialized large cities in western Turkey, the importance of site-specific seismic hazard assessments becomes more crucial. Characterizing source, site and path effects is important for both assessing the seismic hazard in a specific region and generation of the building codes/or renewing previous ones. In this study, we evaluated three-component recordings for micro- and moderate-size earthquakes with local magnitudes ranging between 2.0 and 5.6. This dataset is used for site transfer function estimations, utilizing two different spectral ratio approaches 'Standard Spectral Ratio-(SSR)' and 'Horizontal to Vertical Spectral Ratio-(HVSR)' and a 'Generalized Inversion Technique-(GIT)' to highlight site-specific seismic hazard potential of deep basin structures of the region. Obtained transfer functions revealed that the sites located near the basin edges are characterized by broader HVSR curves. Broad HVSR peaks could be attributed to the complexity of wave propagation related to significant 2D/3D velocity variations at the sediment-bedrock interface near the basin edges. Comparison of HVSR and SSR estimates for the sites located on the grabens showed that SSR estimates give larger values at lower frequencies which could be attributed to lateral variations in regional velocity and attenuation values caused by basin geometry and edge effects. However, large amplitude values of vertical component GIT site transfer functions were observed at varying frequency ranges for some of the stations. These results imply that vertical component of ground motion is not amplification free. Contamination of HVSR site transfer function estimates at different frequency bands could be related to complexities in the wave field caused by deep or shallow heterogeneities in the region such as differences in the basin geometries, fracturing and fluid saturation along

  14. Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

    PubMed Central

    Miletti, Teresa; Di Trani, Justin; Jr Levros, Louis-Charles; Mittermaier, Anthony

    2015-01-01

    Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)+ with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and 15N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity. PMID:25970557

  15. Functionally Important ATP Binding and Hydrolysis Sites in Escherichia coli MsbA †

    PubMed Central

    Westfahl, Kathryn M.; Merten, Jacqueline A.; Buchaklian, Adam H.; Klug, Candice S.

    2009-01-01

    ATP-binding cassette (ABC) transporters make up one of the largest classes of proteins found in nature, and their ability to move a variety of substrates across the membrane using energy from the binding or hydrolysis of ATP is essential to an array of human pathologies and to bacterial viability. MsbA is an essential ABC transporter that specifically transports lipid A across the inner membranes of Gram-negative organisms such as Escherichia coli. The exact mechanisms of function during the binding and hydrolysis of ATP at the molecular level remain unclear. The studies presented and summarized in this work directly address the role and local dynamics of specific residues within the conserved ABC motifs in E. coli MsbA using in vivo growth and biochemical activity assays coupled with site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy motional and accessibility analysis. This first comprehensive analysis of the specific residues in these motifs within MsbA indicates that closure of the dimer interface does not occur upon ATP binding in this transporter. PMID:19053284

  16. A full-potential approach to the relativistic single-site Green's function

    DOE PAGES

    Liu, Xianglin; Wang, Yang; Eisenbach, Markus; ...

    2016-07-07

    One major purpose of studying the single-site scattering problem is to obtain the scattering matrices and differential equation solutions indispensable to multiple scattering theory (MST) calculations. On the other hand, the single-site scattering itself is also appealing because it reveals the physical environment experienced by electrons around the scattering center. In this study, we demonstrate a new formalism to calculate the relativistic full-potential single-site Green's function. We implement this method to calculate the single-site density of states and electron charge densities. Lastly, the code is rigorously tested and with the help of Krein's theorem, the relativistic effects and full potentialmore » effects in group V elements and noble metals are thoroughly investigated.« less

  17. A full-potential approach to the relativistic single-site Green’s function

    NASA Astrophysics Data System (ADS)

    Liu, Xianglin; Wang, Yang; Eisenbach, Markus; Stocks, G. Malcolm

    2016-09-01

    One major purpose of studying the single-site scattering problem is to obtain the scattering matrices and differential equation solutions indispensable to multiple scattering theory (MST) calculations. On the other hand, the single-site scattering itself is also appealing because it reveals the physical environment experienced by electrons around the scattering center. In this paper we demonstrate a new formalism to calculate the relativistic full-potential single-site Green’s function. We implement this method to calculate the single-site density of states and electron charge densities. The code is rigorously tested and with the help of Krein’s theorem, the relativistic effects and full potential effects in group V elements and noble metals are thoroughly investigated.

  18. A full-potential approach to the relativistic single-site Green's function

    SciTech Connect

    Liu, Xianglin; Wang, Yang; Eisenbach, Markus; Stocks, George Malcolm

    2016-07-07

    One major purpose of studying the single-site scattering problem is to obtain the scattering matrices and differential equation solutions indispensable to multiple scattering theory (MST) calculations. On the other hand, the single-site scattering itself is also appealing because it reveals the physical environment experienced by electrons around the scattering center. In this study, we demonstrate a new formalism to calculate the relativistic full-potential single-site Green's function. We implement this method to calculate the single-site density of states and electron charge densities. Lastly, the code is rigorously tested and with the help of Krein's theorem, the relativistic effects and full potential effects in group V elements and noble metals are thoroughly investigated.

  19. Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

    PubMed

    Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

    2016-10-01

    Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ)1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium.

  20. Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli.

    PubMed

    Raynal, A; Tuphile, K; Gerbaud, C; Luther, T; Guérineau, M; Pernodet, J L

    1998-04-01

    The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

  1. Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin

    PubMed Central

    Nobles, Kelly N.; Xiao, Kunhong; Ahn, Seungkirl; Shukla, Arun K.; Lam, Christopher M.; Rajagopal, Sudarshan; Strachan, Ryan T.; Huang, Teng-Yi; Bressler, Erin A.; Hara, Makoto R.; Shenoy, Sudha K.; Gygi, Steven P.; Lefkowitz, Robert J.

    2012-01-01

    Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β2-adrenergic receptor (β2AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β2AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities. PMID:21868357

  2. Quality, Range, and Legibility in Web Sites Related to Orofacial Functions

    PubMed Central

    Corrêa, Camila de Castro; Ferrari, Deborah Viviane; Berretin-Felix, Giédre

    2013-01-01

    Introduction Plenty of information about health is available on the Internet; however, quality and legibility are not always evaluated. Knowledge regarding orofacial functions can be considered important for the population because it allows proper stimulus, early diagnosis, and prevention of the oral myofunctional alterations during early infancy. Objective The aim was evaluate the quality, legibility, and range of Web sites available in Brazilian Portuguese regarding the orofacial functions. Methods Selected Web sites with information directed to parents/caregivers of babies regarding breast-feeding, feeding after 6 months, deleterious oral habits, and breathing and speech were studied. The Web sites were evaluated through the application of Flesch Reading Ease Test and aspects of the Health on the Net (HON) modified code (HONCode); the range of the subjects addressed was compared with other aspects of infant development. Results From the access of 350 pages of the Internet, 35 Web sites were selected and 315 excluded because they did not meet the inclusion criteria. In relation to legibility, Web sites scored an average of 61.23% in the Flesch Test, and the application of the modified HONCode showed an average of 6.43 points; an average of 2.49 subjects were found per Web site evaluated, with information on breast-feeding being more frequent and subjects such as breathing and speech less frequent. Conclusions Web sites that deal with orofacial functions presented standard legibility classification. Only half of the ethical principles were considered by the modified HONCode in their majority, and most addressed subjects after “breast-feeding” were presented with restricted range. PMID:25992036

  3. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism.

    PubMed

    Klingel, U; Miller, C M; North, A K; Stockley, P G; Baumberg, S

    1995-08-21

    In Bacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3' end of an ORF homologous to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 bp AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

  4. 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.

  5. All the catalytic active sites of MoS2 for hydrogen evolution

    DOE PAGES

    Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5more » s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

  6. RNase H Activity: Structure, Specificity, and Function in Reverse Transcription

    PubMed Central

    Schultz, Sharon J.; Champoux, James J.

    2008-01-01

    This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3′ end-directed, and RNA 5′ end-directed. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5′ end-directed and DNA 3′ end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance. PMID:18261820

  7. 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

  8. A novel pattern mining approach for identifying cognitive activity in EEG based functional brain networks.

    PubMed

    Thilaga, M; Vijayalakshmi, R; Nadarajan, R; Nandagopal, D

    2016-06-01

    The complex nature of neuronal interactions of the human brain has posed many challenges to the research community. To explore the underlying mechanisms of neuronal activity of cohesive brain regions during different cognitive activities, many innovative mathematical and computational models are required. This paper presents a novel Common Functional Pattern Mining approach to demonstrate the similar patterns of interactions due to common behavior of certain brain regions. The electrode sites of EEG-based functional brain network are modeled as a set of transactions and node-based complex network measures as itemsets. These itemsets are transformed into a graph data structure called Functional Pattern Graph. By mining this Functional Pattern Graph, the common functional patterns due to specific brain functioning can be identified. The empirical analyses show the efficiency of the proposed approach in identifying the extent to which the electrode sites (transactions) are similar during various cognitive load states.

  9. Improved ethanol electrooxidation performance by shortening Pd–Ni active site distance in Pd–Ni–P nanocatalysts

    PubMed Central

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (∼5 nm) Pd–Ni–P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni–P heterodimers into Pd–Ni–P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites. PMID:28071650

  10. Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts.

    PubMed

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-10

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (∼5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

  11. Improved ethanol electrooxidation performance by shortening Pd-Ni active site distance in Pd-Ni-P nanocatalysts

    NASA Astrophysics Data System (ADS)

    Chen, Lin; Lu, Lilin; Zhu, Hengli; Chen, Yueguang; Huang, Yu; Li, Yadong; Wang, Leyu

    2017-01-01

    Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (~5 nm) Pd-Ni-P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mgPd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni-P heterodimers into Pd-Ni-P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.

  12. 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…

  13. Reconstructing a missing link in the evolution of a recently diverged phosphotriesterase by active-site loop remodeling.

    PubMed

    Afriat-Jurnou, Livnat; Jackson, Colin J; Tawfik, Dan S

    2012-08-07

    Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K(M) values of up to 2 × 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.

  14. Catalytic roles of flexible regions at the active site of ribulose-bisphosphate carboxylase/oxygenase (Rubisco)

    SciTech Connect

    Hartman, F.C.; Harpel, M.R.; Chen, Yuh-Ru; Larson, E.M.; Larimer, F.W.

    1995-12-31

    Chemical and mutagenesis studies of Rubisco have identified Lys329 and Glu48 as active-site residues that are located in distinct, interacting domains from adjacent subunits. Crystallographic analyses have shown that Lys329 is the apical residue in a 12-residue flexible loop (loop 6) of the {Beta},{alpha}-barrel domain of the active site and that Glu48 resides at the end of helix B of the N-terminal domain of the active site. When phosphorylated ligands are bound by the enzyme, loop 6 adopts a closed conformation and, in concert with repositioning of helix B, thereby occludes the active site from the external environment. In this closed conformation, the {gamma}-carboxylate of Glu48 and the {epsilon}-amino group of Lys329 engage in intersubunit electrostatic interaction. By use of appropriate site-directed mutants of Rhodospirillum rubrum Rubisco, we are addressing several issues: the catalytic roles of Lys329 and Glu48, the functional significance of the intersubunit salt bridge comprised of these two residues, and the roles of loop 6 and helix B in stabilizing labile reaction intermediates. Characterization of novel products derived from misprocessing of D-ribulose-1,5-bisphosphate (RuBP) by the mutant proteins have illuminated the structure of the key intermediate in the normal oxygenase pathway.

  15. 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

  16. Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

    PubMed

    Yeung, Juliana H F; Telford, Judith C; Shidmoossavee, Fahimeh S; Bennet, Andrew J; Taylor, Garry L; Moore, Margo M

    2013-12-23

    Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.

  17. Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence, full structure, and active site microenvironment similarity

    PubMed Central

    Leuthaeuser, Janelle B; Knutson, Stacy T; Kumar, Kiran; Babbitt, Patricia C; Fetrow, Jacquelyn S

    2015-01-01

    The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods. PMID:26073648

  18. 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.

  19. Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors.

    PubMed

    Schmitz, Janina; Furtmann, Norbert; Ponert, Moritz; Frizler, Maxim; Löser, Reik; Bartz, Ulrike; Bajorath, Jürgen; Gütschow, Michael

    2015-08-01

    Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values <10 nM. With all dipeptide nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors.

  20. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  1. Barium ions selectively activate BK channels via the Ca2+-bowl site.

    PubMed

    Zhou, Yu; Zeng, Xu-Hui; Lingle, Christopher J

    2012-07-10

    Activation of Ca(2+)-dependent BK channels is increased via binding of micromolar Ca(2+) to two distinct high-affinity sites per BK α-subunit. One site, termed the Ca(2+) bowl, is embedded within the second RCK domain (RCK2; regulator of conductance for potassium) of each α-subunit, while oxygen-containing residues in the first RCK domain (RCK1) have been linked to a separate Ca(2+) ligation site. Although both sites are activated by Ca(2+) and Sr(2+), Cd(2+) selectively favors activation via the RCK1 site. Divalent cations of larger ionic radius than Sr(2+) are thought to be ineffective at activating BK channels. Here we show that Ba(2+), better known as a blocker of K(+) channels, activates BK channels and that this effect arises exclusively from binding at the Ca(2+)-bowl site. Compared with previous estimates for Ca(2+) bowl-mediated activation by Ca(2+), the affinity of Ba(2+) to the Ca(2+) bowl is reduced about fivefold, and coupling of binding to activation is reduced from ∼3.6 for Ca(2+) to about ∼2.8 for Ba(2+). These results support the idea that ionic radius is an important determinant of selectivity differences among different divalent cations observed for each Ca(2+)-binding site.

  2. Structure and Reactivity of the Phosphotriesterase Active Site

    DTIC Science & Technology

    2002-01-01

    characterize different catalytic conformations for chorismate mutase . Preliminary evidence for water binding in phosphotriesterase suggests that activity in...MD/QM study of the chorismate mutase catalyzed Claisen rearrangement reaction. 2001.subm. J.Phys.Chem.B 22.Day, P.N.J., J.H.; Gordon,M.S.; Webb,S.P...Claisen rearrangement of an unusual substrate in chorismate mutase . 2001.subm. J.Phys.Chem.B 38.Stevens, W.J., Basch,H., Krauss,M., Compact effective

  3. Directed Evolution of RebH for Site Selective Halogenation of Large, Biologically Active Molecules**

    PubMed Central

    Payne, James T.; Poor, Catherine B.

    2015-01-01

    We recently characterized the substrate scope of wild-type RebH and evolved variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. We have now used a substrate walking approach to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for site-selective halogenation of biologically active compounds. PMID:25678465

  4. Atomistic characterization of the active-site solvation dynamics of a model photocatalyst

    NASA Astrophysics Data System (ADS)

    van Driel, Tim B.; Kjær, Kasper S.; Hartsock, Robert W.; Dohn, Asmus O.; Harlang, Tobias; Chollet, Matthieu; Christensen, Morten; Gawelda, Wojciech; Henriksen, Niels E.; Kim, Jong Goo; Haldrup, Kristoffer; Kim, Kyung Hwan; Ihee, Hyotcherl; Kim, Jeongho; Lemke, Henrik; Sun, Zheng; Sundström, Villy; Zhang, Wenkai; Zhu, Diling; Møller, Klaus B.; Nielsen, Martin M.; Gaffney, Kelly J.

    2016-11-01

    The interactions between the reactive excited state of molecular photocatalysts and surrounding solvent dictate reaction mechanisms and pathways, but are not readily accessible to conventional optical spectroscopic techniques. Here we report an investigation of the structural and solvation dynamics following excitation of a model photocatalytic molecular system [Ir2(dimen)4]2+, where dimen is para-diisocyanomenthane. The time-dependent structural changes in this model photocatalyst, as well as the changes in the solvation shell structure, have been measured with ultrafast diffuse X-ray scattering and simulated with Born-Oppenheimer Molecular Dynamics. Both methods provide direct access to the solute-solvent pair distribution function, enabling the solvation dynamics around the catalytically active iridium sites to be robustly characterized. Our results provide evidence for the coordination of the iridium atoms by the acetonitrile solvent and demonstrate the viability of using diffuse X-ray scattering at free-electron laser sources for studying the dynamics of photocatalysis.

  5. Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

    PubMed Central

    Haycocks, James R. J.; Grainger, David C.

    2016-01-01

    A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality. PMID:27258043

  6. Functional redundancy of the DE-1 and alpha A-CRYBP1 regulatory sites of the mouse alpha A-crystallin promoter.

    PubMed

    Sax, C M; Ilagan, J G; Piatigorsky, J

    1993-06-11

    Previous studies have implicated the DE-1 (-111/-106) and alpha A-CRYBP1 (-66/-57) sites for activity of the mouse alpha A-crystallin promoter in transiently transfected lens cells. Here we have used the bacterial chloramphenicol acetyltransferase (CAT) reporter gene to test the functional importance of the putative DE-1 and alpha A-CRYBP1 regulatory elements by site-specific and deletion mutagenesis in stably transformed alpha TN4-1 lens cells and in transgenic mice. FVB/N and C57BL/6 x SJL F2 hybrid transgenic mice were assayed for CAT activity in the lens, heart, lung, kidney, spleen, liver, cerebrum, and muscle. F0, F1, and F2 mice from multiple lines carrying single mutations of the DE-1 or alpha A-CRYBP1 sites showed high levels of CAT activity in the lens, but not in any of the non-lens tissues. By contrast, despite activity of the wild-type promoter, none of the mutant promoter/CAT constructs were active in the transiently transfected and stably transformed lens cells. The mice carrying transgenes with either site-specific mutations in both the DE-1 and alpha A-CRYBP1 sites or a deletion of the entire DE-1 and part of the alpha A-CRYBP1 site (-60/+46) fused to the CAT gene did not exhibit CAT activity above background in any of the tissues examined, including the lens. Our results thus indicate that the DE-1 and alpha A-CRYBP1 sites are functionally redundant in transgenic mice. Moreover, the present data coupled with previous transfection and transgenic mouse experiments suggest that this functional redundancy is confined to lens expression within the mouse and is not evident in transiently transfected and stably transformed lens cells, making the cultured lens cells sensitive indicators of functional elements of crystallin genes.

  7. Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.

    PubMed

    Prabhakar, Vikas; Capila, Ishan; Bosques, Carlos J; Pojasek, Kevin; Sasisekharan, Ram

    2005-02-15

    GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

  8. 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.

  9. Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters.

    PubMed Central

    Cromlish, W A; Abmayr, S M; Workman, J L; Horikoshi, M; Roeder, R G

    1989-01-01

    In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements. Images PMID:2539489

  10. A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

    PubMed Central

    Furdui, Cristina; Beamer, Lesa J.

    2012-01-01

    Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues. PMID:22685552

  11. Pathways of H2 toward the Active Site of [NiFe]-Hydrogenase

    PubMed Central

    Teixeira, Vitor H.; Baptista, António M.; Soares, Cláudio M.

    2006-01-01

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2), but little is known about the diffusion of H2 toward the active site. Here we analyze pathways for H2 permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H2 easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H2 molecules that reach the active site made their approach from the side of the Ni ion. H2 is able to reach distances of <4 Å from the active site, although after 6 Å permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H2 inside the protein and at lower distances from the active site. This valine can be a control point in the H2 access to the active center. PMID:16731562

  12. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase.

    PubMed

    O'Brien, P J; Herschlag, D

    2001-05-15

    Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of

  13. A Processive Carbohydrate Polymerase That Mediates Bifunctional Catalysis Using a Single Active Site

    PubMed Central

    May, John F.; Levengood, Matthew R.; Splain, Rebecca A.; Brown, Christopher D.; Kiessling, Laura L.

    2012-01-01

    Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds. PMID:22217153

  14. Structure of protein O-mannose kinase reveals a unique active site architecture

    PubMed Central

    Zhu, Qinyu; Venzke, David; Walimbe, Ameya S; Anderson, Mary E; Fu, Qiuyu; Kinch, Lisa N; Wang, Wei; Chen, Xing; Grishin, Nick V; Huang, Niu; Yu, Liping; Dixon, Jack E; Campbell, Kevin P; Xiao, Junyu

    2016-01-01

    The ‘pseudokinase’ SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome. DOI: http://dx.doi.org/10.7554/eLife.22238.001 PMID:27879205

  15. Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites

    PubMed Central

    Park, Jae-Sook; Thorsness, Mary K.; Policastro, Robert; McGoldrick, Luke L.; Hollingsworth, Nancy M.; Thorsness, Peter E.; Neiman, Aaron M.

    2016-01-01

    The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum–mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome–mitochondrion contacts and to the nuclear–vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc. PMID:27280386

  16. DFT study of the active site of the XoxF-type natural, cerium-dependent methanol dehydrogenase enzyme.

    PubMed

    Bogart, Justin A; Lewis, Andrew J; Schelter, Eric J

    2015-01-19

    Rare-earth metal cations have recently been demonstrated to be essential co-factors for the growth of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. A crystal structure of the rare-earth-dependent methanol dehydrogenase (MDH) includes a cerium cation in the active site. Herein, the Ce-MDH active site has been analyzed through DFT calculations. The results show the stability of the Ce(III)-pyrroloquinoline quinone (PQQ) semiquinone configuration. Calculations on the active oxidized form of this complex indicate a 0.81 eV stabilization of the PQQ(0) LUMO at cerium versus calcium, supporting the observation that the cerium cation in the active site confers a competitive advantage to Methylacidiphilum fumariolicum SolV. Using reported aqueous electrochemical data, a semi-empirical correlation was established based on cerium(IV/III) redox potentials. The correlation allowed estimation of the cerium oxidation potential of +1.35 V versus saturated calomel electrode (SCE) in the active site. The results are expected to guide the design of functional model complexes and alcohol-oxidation catalysts based on lanthanide complexes of biologically relevant quinones.

  17. Parallel sites implicate functional convergence of the hearing gene prestin among echolocating mammals.

    PubMed

    Liu, Zhen; Qi, Fei-Yan; Zhou, Xin; Ren, Hai-Qing; Shi, Peng

    2014-09-01

    Echolocation is a sensory system whereby certain mammals navigate and forage using sound waves, usually in environments where visibility is limited. Curiously, echolocation has evolved independently in bats and whales, which occupy entirely different environments. Based on this phenotypic convergence, recent studies identified several echolocation-related genes with parallel sites at the protein sequence level among different echolocating mammals, and among these, prestin seems the most promising. Although previous studies analyzed the evolutionary mechanism of prestin, the functional roles of the parallel sites in the evolution of mammalian echolocation are not clear. By functional assays, we show that a key parameter of prestin function, 1/α, is increased in all echolocating mammals and that the N7T parallel substitution accounted for this functional convergence. Moreover, another parameter, V1/2, was shifted toward the depolarization direction in a toothed whale, the bottlenose dolphin (Tursiops truncatus) and a constant-frequency (CF) bat, the Stoliczka's trident bat (Aselliscus stoliczkanus). The parallel site of I384T between toothed whales and CF bats was responsible for this functional convergence. Furthermore, the two parameters (1/α and V1/2) were correlated with mammalian high-frequency hearing, suggesting that the convergent changes of the prestin function in echolocating mammals may play important roles in mammalian echolocation. To our knowledge, these findings present the functional patterns of echolocation-related genes in echolocating mammals for the first time and rigorously demonstrate adaptive parallel evolution at the protein sequence level, paving the way to insights into the molecular mechanism underlying mammalian echolocation.

  18. 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

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  19. Sensorimotor Peripheral Nerve Function and Physical Activity in Older Men

    PubMed Central

    Lange-Maia, Brittney S.; Cauley, Jane A.; Newman, Anne B.; Boudreau, Robert M.; Jakicic, John M.; Glynn, Nancy W.; Zivkovic, Sasa; Dam, Tien; Caserotti, Paolo; Cawthon, Peggy M.; Orwoll, Eric S.; Strotmeyer, Elsa S.

    2017-01-01

    We determined whether sensorimotor peripheral nerve (PN) function was associated with physical activity (PA) in older men. The Osteoporotic Fractures in Men Study Pittsburgh, PA, site (n=328, age 78.8±4.7 years), conducted PN testing, including: peroneal motor and sural sensory nerve conduction (latencies, amplitudes: CMAP and SNAP for motor and sensory amplitude, respectively), 1.4g/10g monofilament (dorsum of the great toe), and neuropathy symptoms. ANOVA and multivariate linear regression modeled PN associations with PA (Physical Activity Scale for the Elderly (PASE) and SenseWear Armband). After multivariable adjustment, better motor latency was associated with higher PASE scores (160.5±4.8 vs 135.6±6.7, p<0.01). Those without vs. with neuropathy symptoms had higher PASE scores (157.6±5.3 vs 132.9±7.1, p<0.01). Better vs. worse SNAP was associated with slightly more daily vigorous activity (9.5±0.8 vs. 7.3±0.7, p=0.05). Other PN measures were not associated with PA. Certain PN measures were associated with lower PA, suggesting a potential pathway for disability. PMID:26964668

  20. Constraining Crustal Anisotropy by Receiver Functions at the Deep Continental Drilling Site KTB in Southern Germany

    NASA Astrophysics Data System (ADS)

    Bianchi, Irene; Qorbani, Ehsan; Bokelmann, Götz

    2016-04-01

    As one of the rare observational tools for studying deformation and stress within the Earth, seismic anisotropy has been one of the focuses of geophysical studies over the last decade. In order to unravel the anisotropic properties of the crust, the teleseismic receiver functions (RF) methodology has started to be widely applied recently. Such effects of anisotropy on RF were illustrated in theoretical studies, showing the strong backazimuthal dependence of RF on the 3D characteristics of the media sampled by the waves. The use of teleseismic RF has the advantage of not being affected by a heterogeneous depth distribution of local earthquakes, since teleseismic rays sample the entire crust beneath the stations. The application of this technique however, needs to be critically assessed using a suitable field test. To test the technique, we need a crustal block where the underground structure is reasonably well-known, e.g., where there is extensive knowledge from local seismic experiments and drilling. A field experiment has thus been carried out around the KTB (Kontinental Tiefbohrung) site in the Oberpfalz area in Southeastern Germany, in order to compare with previous results from deep drilling, and high-frequency seismic experiments around the drill site. The investigated region has been studied extensively by local geophysical experiments, and geological studies. The deep borehole was placed into gneiss rocks of the Zone Erbendorf-Vohenstrauss. The drilling activity lasted from 1987 to 1994, and descended down to a depth of 9101 meters, sampling an alternating sequence of paragneiss and amphibolite, with metamorphism of upper amphibolite facies conditions, and ductile deformation produced a strong foliation of the rocks. The application of the RFs reveals strong seismic anisotropy in the upper crust related to the so-called Erbendorf body. The SKS shear-wave splitting method has been applied as well, revealing coherent results for the whole region with exception

  1. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  2. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  3. Fine Control over Site and Substrate Selectivity in Hydrogen Atom Transfer-Based Functionalization of Aliphatic C-H Bonds.

    PubMed

    Salamone, Michela; Carboni, Giulia; Bietti, Massimo

    2016-10-07

    The selective functionalization of unactivated aliphatic C-H bonds over intrinsically more reactive ones represents an ongoing challenge of synthetic chemistry. Here we show that in hydrogen atom transfer (HAT) from the aliphatic C-H bonds of alkane, ether, alcohol, amide, and amine substrates to the cumyloxyl radical (CumO(•)) fine control over site and substrate selectivity is achieved by means of acid-base interactions. Protonation of the amines and metal ion binding to amines and amides strongly deactivates the C-H bonds of these substrates toward HAT to CumO(•), providing a powerful method for selective functionalization of unactivated or intrinsically less reactive C-H bonds. With 5-amino-1-pentanol, site-selectivity has been drastically changed through protonation of the strongly activating NH2 group, with HAT that shifts to the C-H bonds that are adjacent to the OH group. In the intermolecular selectivity studies, trifluoroacetic acid, Mg(ClO4)2, and LiClO4 have been employed in a orthogonal fashion for selective functionalization of alkane, ether, alcohol, and amide (or amine) substrates in the presence of an amine (or amide) one. Ca(ClO4)2, that promotes deactivation of amines and amides by Ca(2+) binding, offers, moreover, the opportunity to selectively functionalize the C-H bonds of alkane, ether, and alcohol substrates in the presence of both amines and amides.

  4. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...). (e) For all asbestos-containing waste material received, the owner or operator of the active waste... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  5. Remarkable difference in catalytic performance of an organoamino-functionalized MCM-41-HPA composite with controlled site-isolation and site-aggregation

    NASA Astrophysics Data System (ADS)

    Chu, Xiaofeng; Le, Ying-Yi; Zhu, Quanjing; Fan, Kangnian; Dai, Wei-Lin

    2011-08-01

    The organoamino-functionalized mesoporous silicas with different distribution patterns—site-isolation or site-aggregation are prepared using post-grafting method. We have investigated the effects of the solvents and the catalytic reactivity of these catalysts. It is found that, using the polar ethanol as solvent, the catalytic center is site-isolated. Contrarily, the catalytic center is site-aggregated with the non-polar toluene. Characterization techniques, including transmission electron microscopy, nitrogen sorption experiments, thermogravimetric analysis, and ultraviolet-visible absorbance spectroscopy, demonstrate the most important dependencies of the distribution pattern on the polarity of solvent.

  6. Structure and nuclearity of active sites in Fe-zeolites: comparison with iron sites in enzymes and homogeneous catalysts.

    PubMed

    Zecchina, Adriano; Rivallan, Mickaël; Berlier, Gloria; Lamberti, Carlo; Ricchiardi, Gabriele

    2007-07-21

    Fe-ZSM-5 and Fe-silicalite zeolites efficiently catalyse several oxidation reactions which find close analogues in the oxidation reactions catalyzed by homogeneous and enzymatic compounds. The iron centres are highly dispersed in the crystalline matrix and on highly diluted samples, mononuclear and dinuclear structures are expected to become predominant. The crystalline and robust character of the MFI framework has allowed to hypothesize that the catalytic sites are located in well defined crystallographic positions. For this reason these catalysts have been considered as the closest and best defined heterogeneous counterparts of heme and non heme iron complexes and of Fenton type Fe(2+) homogeneous counterparts. On this basis, an analogy with the methane monooxygenase has been advanced several times. In this review we have examined the abundant literature on the subject and summarized the most widely accepted views on the structure, nuclearity and catalytic activity of the iron species. By comparing the results obtained with the various characterization techniques, we conclude that Fe-ZSM-5 and Fe-silicalite are not the ideal samples conceived before and that many types of species are present, some active and some other silent from adsorptive and catalytic point of view. The relative concentration of these species changes with thermal treatments, preparation procedures and loading. Only at lowest loadings the catalytically active species become the dominant fraction of the iron species. On the basis of the spectroscopic titration of the active sites by using NO as a probe, we conclude that the active species on very diluted samples are isolated and highly coordinatively unsaturated Fe(2+) grafted to the crystalline matrix. Indication of the constant presence of a smaller fraction of Fe(2+) presumably located on small clusters is also obtained. The nitrosyl species formed upon dosing NO from the gas phase on activated Fe-ZSM-5 and Fe-silicalite, have been analyzed

  7. Structural and functional studies of CCAAT/enhancer binding sites within the human immunodeficiency type 1 subtype C LTR

    PubMed Central

    Liu, Yujie; Nonnemacher, Michael R.; Stauff, Devin L.; Li, Luna; Banerjee, Anupam; Irish, Bryan; Kilareski, Evelyn; Rajagopalan, Nirmala; Suchitra, Joyce B.; Khan, Zafar K.; Ranga, Udaykumar; Wigdahl, Brian

    2010-01-01

    Human immunodeficiency virus type 1 (HIV-1) subtype C, which is most predominant in sub-Saharan Africa as well as in Asia and India, is the most prevalent subtype worldwide. A large number of transcription factor families have been shown to be involved in regulating HIV-1 gene expression in T lymphocytes and cells of the monocyte-macrophage lineage. Among these, proteins of the CCAAT/enhancer binding protein (C/EBP) family are of particular importance in regulating HIV-1 gene expression within cells of the monocytic lineage during the course of hematologic development and cellular activation. Few studies have examined the role of C/EBPs in long terminal repeat (LTR)-directed viral gene expression of HIV-1 subtypes other than subtype B. Within subtype B viruses, two functional C/EBP sites located upstream of the TATA box are required for efficient viral replication in cells of the monocyte-macrophage lineage. We report the identification of three putative subtype C C/EBP sites, upstream site 1 and 2 (C-US1 and C-US2) and downstream site 1 (C-DS1). C-US1 and C-DS1 were shown to form specific DNA-protein complexes with members of the C/EBP family (C/EBPα, β, and δ). Functionally, within the U-937 monocytic cell line, subtype B and C LTRs were shown to be equally responsive to C/EBPβ-2, although the basal activity of subtype C LTRs appeared to be higher. Furthermore, the synergistic interaction between C/EBPβ-2 and Tat with the subtype C LTR was also observed in U-937 cells as previously demonstrated with the subtype B LTR. PMID:20970301

  8. The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes.

    PubMed

    Kunze, K L; Mangold, B L; Wheeler, C; Beilan, H S; Ortiz de Montellano, P R

    1983-04-10

    Hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats is inactivated during the metabolism of linear olefins (ethylene, propene, and octene) and acetylenes (acetylene, propyne, and octyne). As expected from previous work, the inactivation is due to N-alkylation of the prosthetic heme group by the substrate. The N-alkyl group in each adduct is formally obtained by addition of a porphyrin nitrogen to the terminal carbon and of an oxygen atom (as a hydroxyl function) to the internal carbon of the pi-bond. The oxygen is shown here by 18O studies to be catalytically introduced by the enzyme. The olefins exclusively alkylate the nitrogen of pyrrole ring D, but the acetylenes alkylate that of pyrrole ring A. Acetylene is an exception in that it reacts with more than one nitrogen. Circular dichroism studies of the ethylene adduct and of the ring D regioisomer of N-ethylprotoporphyrin IX obtained by alkylation of the prosthetic heme of hemoglobin have been used to determine which face of cytochrome P-450 heme is alkylated by the unsaturated substrates. These results implicate an active site that is sterically encumbered in the region over pyrrole ring B and has a lipophilic binding site that accommodates chains of at least six carbon atoms over pyrrole ring C.

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

    DOE PAGES

    Wang, Yanggang; Mei, Donghai; Glezakou, Vassiliki Alexandra; ...

    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

  10. 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.

  11. Microbial community changes along the active seepage site of one cold seep in the Red Sea

    PubMed Central

    Cao, Huiluo; Zhang, Weipeng; Wang, Yong; Qian, Pei-Yuan

    2015-01-01

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep. PMID:26284035

  12. Microbial community changes along the active seepage site of one cold seep in the Red Sea.

    PubMed

    Cao, Huiluo; Zhang, Weipeng; Wang, Yong; Qian, Pei-Yuan

    2015-01-01

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep.

  13. The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

    PubMed

    Cottrell, Joseph W; Scott, Lincoln G; Fedor, Martha J

    2011-05-20

    Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

  14. Direct Visualization of Catalytically Active Sites at the FeO-Pt(111) Interface

    SciTech Connect

    Kudernatsch, Wilhelmine; Peng, Guowen; Zeuthen, Helene; Bai, Yunhai; Merte, L. R.; Lammich, Lutz; Besenbacher, Fleming; Mavrikakis, Manos; Wendt, Stefen

    2015-08-25

    Within the area of surface science, one of the “holy grails” is to directly visualize a chemical reaction at the atomic scale. Whereas this goal has been reached by high-resolution scanning tunneling microscopy (STM) in a number of cases for reactions occurring at flat surfaces, such a direct view is often inhibited for reaction occurring at steps and interfaces. Here we have studied the CO oxidation reaction at the interface between ultrathin FeO islands and a Pt(111) support by in situ STM and density functional theory (DFT) calculations. Time-lapsed STM imaging on this inverse model catalyst in O2 and CO environments revealed catalytic activity occurring at the FeO-Pt(111) interface and directly showed that the Fe-edges host the catalytically most active sites for the CO oxidation reaction. This is an important result since previous evidence for the catalytic activity of the FeO-Pt(111) interface is essentially based on averaging techniques in conjunction with DFT calculations. The presented STM results are in accord with DFTþU calculations, in which we compare possible CO oxidation pathways on oxidized Fe-edges and O-edges. We found that the CO oxidation reaction is more favorable on the oxidized Fe-edges, both thermodynamically and kinetically.

  15. Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

    PubMed

    Sebti, Y; Rabbani, M; Sadeghi, H Mir Mohammad; Sardari, S; Ghahremani, M H; Innamorati, G

    2015-01-01

    The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

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

    SciTech Connect

    Not Available

    1994-02-01

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

  17. Functional link between surface low-coordination sites and the electrochemical durability of Pt nanoparticles

    NASA Astrophysics Data System (ADS)

    Chung, Dong Young; Shin, Heejong; Yoo, Ji Mun; Lee, Kug-Seung; Lee, Nam-Suk; Kang, Kisuk; Sung, Yung-Eun

    2016-12-01

    A promising strategy for achieving enhanced catalytic activity involves the use of nanoscale electrocatalysts; however, their low stability remains a major challenge. Among the various performance-degradation mechanisms, atomic dissolution is known to cause severe nanoparticle deactivation. To date, the factors influencing these catalysts' durability are not understood. Herein, we assess the role of low-coordination surface sites, focusing on the atomic dissolution of Pt nanoparticles. The density of low-coordination sites was finely controlled, and no significant size change occurred. Based on our findings, we suggest that the initial low-coordination sites trigger metal dissolution, which subsequently accelerates Pt dissolution. We believe that controlling the surface coordination number can open new routes for the design of highly durable nanoscale electrocatalysts.

  18. Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH.

    PubMed

    Suno, Ryoji; Shimoyama, Masakazu; Abe, Akiko; Shimamura, Tatsuro; Shimodate, Natsuka; Watanabe, Yo-hei; Akiyama, Yoshinori; Yoshida, Masasuke

    2012-09-21

    When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

  19. Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Garcia-Horsman, J Arturo; Wallén, Erik A A; Christiaans, Johannes A M; Jarho, Elina M; Gynther, Jukka; Männistö, Pekka T

    2004-09-15

    POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH(2)OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (K(i)=11.8-0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, k(on)=(2.43+/-0.12) x 10(5) M(-1) x s(-1), whereas the inhibitor with a CN group exhibited the fastest binding, k(on)=(12.0+/-0.08)x10(5) M(-1) x s(-1). In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH(2)OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the K(i) values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.

  20. Site-specific functionalization of proteins and their applications to therapeutic antibodies

    PubMed Central

    van Vught, Remko; Pieters, Roland J; Breukink, Eefjan

    2014-01-01

    Protein modifications are often required to study structure and function relationships. Instead of the random labeling of lysine residues, methods have been developed to (sequence) specific label proteins. Next to chemical modifications, tools to integrate new chemical groups for bioorthogonal reactions have been applied. Alternatively, proteins can also be selectively modified by enzymes. Herein we review the methods available for site-specific modification of proteins and their applications for therapeutic antibodies. PMID:24757499

  1. Crystal structure at 1.63 A resolution of the native form of porcine beta-trypsin: revealing an acetate ion binding site and functional water network.

    PubMed

    Johnson, A; Gautham, N; Pattabhi, V

    1999-11-16

    The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine beta-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 A resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-HellipsisO hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cbeta-Ogamma-His-57 Nepsilon angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.

  2. The surface chemistry of heterogeneous catalysis: mechanisms, selectivity, and active sites.

    PubMed

    Zaera, Francisco

    2005-01-01

    The role of chemical kinetics in defining the requirements for the active sites of heterogeneous catalysts is discussed. A personal view is presented, with specific examples from our laboratory to illustrate the role of the chemical composition, structure, and electronic properties of specific surface sites in determining reaction activity and selectivity. Manipulation of catalytic behavior via the addition of chemical modifiers and by tuning of the reaction conditions is also introduced.

  3. Bidentate ligation of heme analogues; novel biomimetics of peroxidase active site.

    PubMed

    Ashkenasy, Gonen; Margulies, David; Felder, Clifford E; Shanzer, Abraham; Powers, Linda S

    2002-09-02

    The multifunctional nature of proteins that have iron-heme cofactors with noncovalent histidine linkage to the protein is controlled by the heme environment. Previous studies of these active-site structures show that the primary difference is the length of the iron-proximal histidine bond, which can be controlled by the degree of H-bonding to this histidine. Great efforts to mimic these functions with synthetic analogues have been made for more than two decades. The peroxidase models resulted in several catalytic systems capable of a large range of oxidative transformations. Most of these model systems modified the porphyrin ring covalently by directly binding auxiliary elements that control and facilitate reactivity; for example, electron-donating or -withdrawing substituents. A biomimetic approach to enzyme mimicking would have taken a different route, by attempting to keep the porphyrin ring system unaltered, as close as possible to its native form, and introducing all modifications at or close to the axial coordination sites. Such a model system would be less demanding synthetically, would make it easy to study the effect of a single structural modification, and might even provide a way to probe effects resulting from porphyrin exchange. We introduce here an alternative model system based on these principles. It consists of a two component system: a bis-imidazolyl ligand and an iron-porphyrin (readily substituted by a hemin). All modifications were introduced only to the ligand that engulfs the porphyrin and binds to the iron's fifth and sixth coordination sites. We describe the design, synthesis, and characterization of nine different model compounds with increased complexity. The primary tool for characterizing the environment of each complex Fe(III) center was the Extended X-ray Absorption Fine Structure (EXAFS) measurements, supported by UV/Vis, IR, and NMR spectroscopy and by molecular modeling. Introduction of asymmetry, by attaching different imidazoles

  4. Nuclear waste: Status of DOE`s nuclear waste site characterization activities

    SciTech Connect

    1987-12-31

    Three potential nuclear waste repository sites have been selected to carry out characterization activities-the detailed geological testing to determine the suitability of each site as a repository. The sites are Hanford in south-central Washington State, Yucca Mountain in southern Nevada, and Deaf Smith in the Texas Panhandle. Two key issues affecting the total program are the estimations of the site characterization completion data and costs and DOE`s relationship with the Nuclear Regulatory Commission which has been limited and its relations with affected states and Indian tribes which continue to be difficult.

  5. Functional microRNAs and target sites are created by lineage-specific transposition

    PubMed Central

    Spengler, Ryan M.; Oakley, Clayton K.; Davidson, Beverly L.

    2014-01-01

    Transposable elements (TEs) account for nearly one-half of the sequence content in the human genome, and de novo germline transposition into regulatory or coding sequences of protein-coding genes can cause heritable disorders. TEs are prevalent in and around protein-coding genes, providing an opportunity to impart regulation. Computational studies reveal that microRNA (miRNA) genes and miRNA target sites reside within TE sequences, but there is little experimental evidence supporting a role for TEs in the birth of miRNAs, or as platform for gene regulation by miRNAs. In this work, we validate miRNAs and target sites derived from TE families prevalent in the human genome, including the ancient long interspersed nuclear element 2 (LINE2/L2), mammalian-wide interspersed repeat (MIR) retrotransposons and the primate-specific Alu family. We show that genes with 3′ untranslated region (3′ UTR) MIR elements are enriched for let-7 targets and that these sites are conserved and responsive to let-7 expression. We also demonstrate that 3′ UTR-embedded Alus are a source of miR-24 and miR-122 target sites and that a subset of active genomic Alus provide for de novo target site creation. Finally, we report that although the creation of miRNA genes by Alu elements is relatively uncommon relative to their overall genomic abundance, Alu-derived miR-1285-1 is efficiently processed from its genomic locus and regulates genes with target sites contained within homologous elements. Taken together, our data provide additional evidence for TEs as a source for miRNAs and miRNA target sites, with instances of conservation through the course of mammalian evolution. PMID:24234653

  6. Functional microRNAs and target sites are created by lineage-specific transposition.

    PubMed

    Spengler, Ryan M; Oakley, Clayton K; Davidson, Beverly L

    2014-04-01

    Transposable elements (TEs) account for nearly one-half of the sequence content in the human genome, and de novo germline transposition into regulatory or coding sequences of protein-coding genes can cause heritable disorders. TEs are prevalent in and around protein-coding genes, providing an opportunity to impart regulation. Computational studies reveal that microRNA (miRNA) genes and miRNA target sites reside within TE sequences, but there is little experimental evidence supporting a role for TEs in the birth of miRNAs, or as platform for gene regulation by miRNAs. In this work, we validate miRNAs and target sites derived from TE families prevalent in the human genome, including the ancient long interspersed nuclear element 2 (LINE2/L2), mammalian-wide interspersed repeat (MIR) retrotransposons and the primate-specific Alu family. We show that genes with 3' untranslated region (3' UTR) MIR elements are enriched for let-7 targets and that these sites are conserved and responsive to let-7 expression. We also demonstrate that 3' UTR-embedded Alus are a source of miR-24 and miR-122 target sites and that a subset of active genomic Alus provide for de novo target site creation. Finally, we report that although the creation of miRNA genes by Alu elements is relatively uncommon relative to their overall genomic abundance, Alu-derived miR-1285-1 is efficiently processed from its genomic locus and regulates genes with target sites contained within homologous elements. Taken together, our data provide additional evidence for TEs as a source for miRNAs and miRNA target sites, with instances of conservation through the course of mammalian evolution.

  7. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  8. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    PubMed Central

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

  9. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal–Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems

    PubMed Central

    2015-01-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe–Nx sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e– × 2e– mechanism in alkaline media on the primary Fe2+–N4 centers and the dual-site 2e– × 2e– mechanism in acid media with the significant role of the surface bound coexisting Fe/FexOy nanoparticles (NPs) as the secondary active sites. PMID:24817921

  10. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    NASA Astrophysics Data System (ADS)

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

  11. Elucidating Oxygen Reduction Active Sites in Pyrolyzed Metal-Nitrogen Coordinated Non-Precious-Metal Electrocatalyst Systems.

    PubMed

    Tylus, Urszula; Jia, Qingying; Strickland, Kara; Ramaswamy, Nagappan; Serov, Alexey; Atanassov, Plamen; Mukerjee, Sanjeev

    2014-05-01

    Detailed understanding of the nature of the active centers in non-precious-metal-based electrocatalyst, and their role in oxygen reduction reaction (ORR) mechanistic pathways will have a profound effect on successful commercialization of emission-free energy devices such as fuel cells. Recently, using pyrolyzed model structures of iron porphyrins, we have demonstrated that a covalent integration of the Fe-N x sites into π-conjugated carbon basal plane modifies electron donating/withdrawing capability of the carbonaceous ligand, consequently improving ORR activity. Here, we employ a combination of in situ X-ray spectroscopy and electrochemical methods to identify the various structural and functional forms of the active centers in non-heme Fe/N/C catalysts. Both methods corroboratively confirm the single site 2e(-) × 2e(-) mechanism in alkaline media on the primary Fe(2+)-N4 centers and the dual-site 2e(-) × 2e(-) mechanism in acid media with the significant role of the surface bound coexisting Fe/Fe x O y nanoparticles (NPs) as the secondary active sites.

  12. Logic minimization and rule extraction for identification of functional sites in molecular sequences

    PubMed Central

    2012-01-01

    Background Logic minimization is the application of algebraic axioms to a binary dataset with the purpose of reducing the number of digital variables and/or rules needed to express it. Although logic minimization techniques have been applied to bioinformatics datasets before, they have not been used in classification and rule discovery problems. In this paper, we propose a method based on logic minimization to extract predictive rules for two bioinformatics problems involving the identification of functional sites in molecular sequences: transcription factor binding sites (TFBS) in DNA and O-glycosylation sites in proteins. TFBS are important in various developmental processes and glycosylation is a posttranslational modification critical to protein functions. Methods In the present study, we first transformed the original biological dataset into a suitable binary form. Logic minimization was then applied to generate sets of simple rules to describe the transformed dataset. These rules were used to predict TFBS and O-glycosylation sites. The TFBS dataset is obtained from the TRANSFAC database, while the glycosylation dataset was compiled using information from OGLYCBASE and the Swiss-Prot Database. We performed the same predictions using two standard classification techniques, Artificial Neural Networks (ANN) and Support Vector Machines (SVM), and used their sensitivities and positive predictive values as benchmarks for the performance of our proposed algorithm. SVM were also used to reduce the number of variables included in the logic minimization approach. Results For both TFBS and O-glycosylation sites, the prediction performance of the proposed logic minimization method was generally comparable and, in some cases, superior to the standard ANN and SVM classification methods with the advantage of providing intelligible rules to describe the datasets. In TFBS prediction, logic minimization produced a very small set of simple rules. In glycosylation site prediction

  13. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  14. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  15. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  16. Identification of a putative binding site critical for general anesthetic activation of TRPA1.

    PubMed

    Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

    2017-04-04

    General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

  17. Modeling injury rates as a function of industrialized versus on-site construction techniques.

    PubMed

    Rubio-Romero, J C; Suárez-Cebador, M; Abad, Jesús

    2014-05-01

    It is often predicted that the industrialization of building activities will lead to a reduction of accident rates in the construction sector, particularly as a result of switching activities from building sites to factories. However, to date no scientific research has provided objective quantitative results to back up this claim. The aim of this paper is to evaluate how industrialization affects the accident rate in different industrialized building systems in Spain. Our results revealed that the industrialized steel modular system presents the lowest accident rate, while the highest accident rate was recorded in the construction method with cast-in-place concrete. The lightweight construction system also presents a high accident rate. Accordingly, industrialized building systems cannot claim to be safer than traditional ones. The different types of "on-site work" seem to be the main variable which would explain the accident rates recorded in industrialized construction systems.

  18. Predicting diffuse light-enhancement of GPP from plant functional traits: A multi-site synthesis

    NASA Astrophysics Data System (ADS)

    O'Halloran, T. L.; Barr, J. G.; Cook, B.; Goeckede, M.; Law, B. E.; Kueppers, L. M.; Riley, W. J.

    2013-12-01

    Diffuse light enhances canopy-scale photosynthesis because isotropic diffuse light penetrates deeper into the canopy, involves more leaf area in photosynthesis, and prevents the top of the canopy from becoming light saturated. However, the observational and modeling communities still have little understanding of how the 'Diffuse light Enhancement Effect' (DEE) varies across plant functional types or is constrained by factors such as nitrogen availability and plant structure. So far, variability in the strength of DEE across plant functional types (PFTs) remains poorly constrained, but canopy models indicate leaf area index (LAI) is a primary controller. While the very few existing multi-site, measurement-based syntheses of the DEE have provided valuable information on the variability of the DEE across a few plant functional types, no study has correlated measured metrics of DEE magnitude with direct measurements of canopy physical traits across a wide range of plant functional types. Here we report a new metric that is suitable for quantifying the DEE in both flux measurements and land surface models. We also present, for the first time, an examination of the relationship between the DEE metric and plant functional traits. Results from our 70+ site AmeriFlux and FLUXNET synthesis indicate that LAI is the strongest controller of the DEE across sites and PFTs, with less significant influences from foliar nitrogen, canopy height, and mean annual precipitation. Our results will enable direct evaluation and improvement of remote sensing algorithms and light use efficiency models (e.g. MODIS GPP), which to this point regard diffuse light fraction as a source of noise. Additionally, improving resolution of the DEE in prognostic land surface models, such as the Community Land Model (CLM), will greatly improve our ability to forecast future feedbacks to terrestrial carbon sequestration from changes in cloudiness and aerosol amount.

  19. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    PubMed

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.

  20. Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies.

    PubMed

    Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

    2011-06-01

    The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

  1. Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

    NASA Astrophysics Data System (ADS)

    Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

    2011-06-01

    The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

  2. Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design

    PubMed Central

    Saravanamuthu, Ahilan; Vickers, Tim J.; Bond, Charles S.; Peterson, Mark R.; Hunter, William N.; Fairlamb, Alan H.

    2012-01-01

    SUMMARY Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible. PMID:15102853

  3. The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

    PubMed

    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

    2014-09-05

    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 Pro(216)-Phe(228) 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.

  4. Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

    PubMed Central

    Swale, Daniel R.; Sheehan, Jonathan H.; Banerjee, Sreedatta; Husni, Afeef S.; Nguyen, Thuy T.; Meiler, Jens; Denton, Jerod S.

    2015-01-01

    The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val168 or Asn171 in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK. PMID:25762321

  5. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.

  6. All the catalytic active sites of MoS2 for hydrogen evolution

    SciTech Connect

    Li, Guoqing; Zhang, Du; Qiao, Qiao; Yu, Yifei; Peterson, David; Zafar, Abdullah; Kumar, Raj; Curtarolo, Stefano; Hunte, Frank; Shannon, Steve; Zhu, Yimei; Yang, Weitao; Cao, Linyou

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5 s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.

  7. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  8. Developing restoration planting mixes for active ski slopes: a multi-site reference community approach.

    PubMed

    Burt, Jennifer Williamson

    2012-03-01

    Downhill ski areas occupy large expanses of mountainous lands where restoration of ecosystem function is of increasing importance and interest. Establishing diverse native plant communities on ski runs should enhance sediment and water retention, wildlife habitat, biodiversity and aesthetics. Because ski slopes are managed for recreation, ski slope revegetation mixes must consist of low-stature or herbaceous plants that can tolerate typical environmental conditions on ski slopes (high elevation, disturbed soils, open, steep slopes). The most appropriate reference communities for selecting ski slope revegetation species are thus successional, or seral plant communities in similar environments (i.e., other ski slopes). Using results from a broad-scale reference community analysis, I evaluated plant communities naturally occurring on ski slopes from 21 active and abandoned ski areas throughout the northern Sierra Nevada to identify native plant species suitable for use in ski slope restoration. I constructed a baseline planting palette of regionally appropriate plant species (for restoration of either newly created or already existing ski runs) that is functionally diverse and is likely to succeed across a broad range of environments. I also identify a more comprehensive list of species for more specialized planting mixes based on site-specific goals and particular environmental settings. Establishing seral plant communities may be an appropriate restoration goal for many other types of managed lands, including roadsides, firebreaks and utility rights-of-way. This study describes an ecological (and potentially cost-effective) approach to developing restoration planting palettes for such managed lands.

  9. Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response.

    PubMed

    Hazan, Idit; Hofmann, Thomas G; Aqeilan, Rami I

    2016-12-01

    The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells.

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

    SciTech Connect

    Jennings, L.; Bohon, J; Chance, M; Licht, S

    2008-01-01

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

  11. Developing Restoration Planting Mixes for Active Ski Slopes: A Multi-Site Reference Community Approach

    NASA Astrophysics Data System (ADS)

    Burt, Jennifer Williamson

    2012-03-01

    Downhill ski areas occupy large expanses of mountainous lands where restoration of ecosystem function is of increasing importance and interest. Establishing diverse native plant communities on ski runs should enhance sediment and water retention, wildlife habitat, biodiversity and aesthetics. Because ski slopes are managed for recreation, ski slope revegetation mixes must consist of low-stature or herbaceous plants that can tolerate typical environmental conditions on ski slopes (high elevation, disturbed soils, open, steep slopes). The most appropriate reference communities for selecting ski slope revegetation species are thus successional, or seral plant communities in similar environments (i.e., other ski slopes). Using results from a broad-scale reference community analysis, I evaluated plant communities naturally occurring on ski slopes from 21 active and abandoned ski areas throughout the northern Sierra Nevada to identify native plant species suitable for use in ski slope restoration. I constructed a baseline planting palette of regionally appropriate plant species (for restoration of either newly created or already existing ski runs) that is functionally diverse and is likely to succeed across a broad range of environments. I also identify a more comprehensive list of species for more specialized planting mixes based on site-specific goals and particular environmental settings. Establishing seral plant communities may be an appropriate restoration goal for many other types of managed lands, including roadsides, firebreaks and utility rights-of-way. This study describes an ecological (and potentially cost-effective) approach to developing restoration planting palettes for such managed lands.

  12. Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response

    PubMed Central

    Hazan, Idit; Hofmann, Thomas G.; Aqeilan, Rami I.

    2016-01-01

    The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one suggests that CFS loci are hotspots of genomic instability leading to inactivation of genes encoded within them, while the other view proposes that CFSs are functional units and that loss of the encoded genes confers selective pressure, leading to cancer development. The latter view is supported by emerging evidence showing that expression of a given CFS is associated with genome integrity and that inactivation of CFS-resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two viewpoints of CFS function are not mutually exclusive but rather coexist; when breaks at CFSs are not repaired accurately, this can lead to deletions by which cells acquire growth advantage because of loss of tumor suppressor activities. Here, we review recent advances linking some CFS gene products with the DDR, genomic instability, and carcinogenesis and discuss how their inactivation might represent a selective advantage for cancer cells. PMID:27977694

  13. Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability.

    PubMed

    Ong, Chin-Tong; Cheng, Hui-Teng; Chang, Li-Wei; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Stormo, Gary D; Kopan, Raphael

    2006-02-24

    All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jkappa), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jkappa-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that "preferred" targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

  14. Is there a need for site productivity functions for short-rotation woody crop plantings?

    SciTech Connect

    Downing, M.; Tuskan, G.A.

    1995-07-06

    For over a decade, researchers have used small-scale research plots to assist development and selection of high yielding, pest-resistant clones of fast-growing hardwoods such as hybrid poplar (Populus spp.). Substantial advances have been made in the techniques and criteria for screening species and selecting clones. Data from these research plots indicate that the ultimate performance of selected clones is dependent upon variable factors in the environment. Until now, researchers could only determine the suitability of a given site for such clones, not the actual yield potential of the site. Recently in the north central US, several clones were planted on larger-than-research-scale plots on private land recontracted under the Conservation Reserve Program (CRP). The historical database could not provide a framework which would allow producers to predict the yield potential of a particular clone on a specific site. Through a systematic combination of clonal trials on experimental research-scale plots and operational plantings on 50 to 100 acre agricultural-scale field plots, it may be possible to develop yield functions or site quality equations which would predict biomass yields at rotation for selected clones. Such estimates will (1) reduce the probability of planting failure, (2) allow maximum expression of the genetic potential of selected superior clones, and thus (3) facilitate accurate economic planning for both the producer and conversion facility manager.

  15. Modeling and structure function analysis of the putative anchor site of yeast telomerase

    PubMed Central

    Lue, Neal F.; Li, Zhaohui

    2007-01-01

    Telomerase is a ribonucleoprotein reverse transcriptase responsible for extending one strand of the telomere terminal repeats. Unique among reverse transcriptases, telomerase is thought to possess a DNA-binding domain (known as anchor site) that allows the enzyme to add telomere repeats processively. Previous crosslinking and mutagenesis studies have mapped the anchor site to an N-terminal region of TERT, and the structure of this region of Tetrahymena TERT was recently determined at atomic resolutions. Here we use a combination of homology modeling, electrostatic calculation and site-specific mutagenesis analysis to identify a positively charged, functionally important surface patch on yeast TERT. This patch is lined by both conserved and non-conserved residues, which when mutated, caused loss of telomerase processivity in vitro and telomere shortening in vivo. In addition, we demonstrate that a point mutation in this domain of yeast TERT simultaneously enhanced the repeat addition processivity of telomerase and caused telomere elongation. Our data argue that telomerase anchor site has evolved species-specific residues to interact with species-specific telomere repeats. The data also reinforce the importance of telomerase processivity in regulating telomere length. PMID:17670795

  16. Extraction of Functional Binding Sites from Unique Regulatory Regions: The Drosophila Early Developmental Enhancers

    PubMed Central

    Papatsenko, Dmitri A.; Makeev, Vsevolod J.; Lifanov, Alex P.; Régnier, Mireille; Nazina, Anna G.; Desplan, Claude

    2002-01-01

    The early developmental enhancers of Drosophila melanogaster comprise one of the most sophisticated regulatory systems in higher eukaryotes. An elaborate code in their DNA sequence translates both maternal and early embryonic regulatory signals into spatial distribution of transcription factors. One of the most striking features of this code is the redundancy of binding sites for these transcription factors (BSTF). Using this redundancy, we explored the possibility of predicting functional binding sites in a single enhancer region without any prior consensus/matrix description or evolutionary sequence comparisons. We developed a conceptually simple algorithm, Scanseq, that employs an original statistical evaluation for identifying the most redundant motifs and locates the position of potential BSTF in a given regulatory region. To estimate the biological relevance of our predictions, we built thorough literature-based annotations for the best-known Drosophila developmental enhancers and we generated detailed distribution maps for the most robust binding sites. The high statistical correlation between the location of BSTF in these experiment-based maps and the location predicted in silico by Scanseq confirmed the relevance of our approach. We also discuss the definition of true binding sites and the possible biological principles that govern patterning of regulatory regions and the distribution of transcriptional signals. PMID:11875036

  17. Active sites and mechanisms for H₂O₂ decomposition over Pd catalysts.

    PubMed

    Plauck, Anthony; Stangland, Eric E; Dumesic, James A; Mavrikakis, Manos

    2016-04-05

    A combination of periodic, self-consistent density functional theory (DFT-GGA-PW91) calculations, reaction kinetics experiments on a SiO2-supported Pd catalyst, and mean-field microkinetic modeling are used to probe key aspects of H2O2 decomposition on Pd in the absence of cofeeding H2 We conclude that both Pd(111) and OH-partially covered Pd(100) surfaces represent the nature of the active site for H2O2 decomposition on the supported Pd catalyst reasonably well. Furthermore, all reaction flux in the closed catalytic cycle is predicted to flow through an O-O bond scission step in either H2O2 or OOH, followed by rapid H-transfer steps to produce the H2O and O2 products. The barrier for O-O bond scission is sensitive to Pd surface structure and is concluded to be the central parameter governing H2O2 decomposition activity.

  18. Active sites and mechanisms for H2O2 decomposition over Pd catalysts

    PubMed Central

    Plauck, Anthony; Stangland, Eric E.; Dumesic, James A.; Mavrikakis, Manos

    2016-01-01

    A combination of periodic, self-consistent density functional theory (DFT-GGA-PW91) calculations, reaction kinetics experiments on a SiO2-supported Pd catalyst, and mean-field microkinetic modeling are used to probe key aspects of H2O2 decomposition on Pd in the absence of cofeeding H2. We conclude that both Pd(111) and OH-partially covered Pd(100) surfaces represent the nature of the active site for H2O2 decomposition on the supported Pd catalyst reasonably well. Furthermore, all reaction flux in the closed catalytic cycle is predicted to flow through an O–O bond scission step in either H2O2 or OOH, followed by rapid H-transfer steps to produce the H2O and O2 products. The barrier for O–O bond scission is sensitive to Pd surface structure and is concluded to be the central parameter governing H2O2 decomposition activity. PMID:27006504

  19. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

    PubMed Central

    Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

  20. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  1. Measurement of functional activities in older adults in the community.

    PubMed

    Pfeffer, R I; Kurosaki, T T; Harrah, C H; Chance, J M; Filos, S

    1982-05-01

    Two measures of social function designed for community studies of normal aging and mild senile dementia were evaluated in 195 older adults who underwent neurological, cognitive, and affective assessment. An examining and a reviewing neurologist and a neurologically trained nurse independently rated each on a Scale of Functional Capacity. Interrater reliability was high (examining vs. reviewing neurologist, r = .97; examining neurologist vs. nurse, tau b = .802; p less than .001 for both comparisons). Estimates correlated well with an established measure of social function and with results of cognitive tests. Alternate informants evaluated participants on the Functional Activities Questionnaire and the Instrumental Activities of Daily Living Scale. The Functional Activities Questionnaire was superior to the Instrumental Activities of Daily scores. Used alone as a diagnostic tool, the Functional Activities Questionnaire was more sensitive than distinguishing between normal and demented individuals.

  2. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  3. Site-resolved measurement of the spin-correlation function in the Fermi-Hubbard model

    NASA Astrophysics Data System (ADS)

    Parsons, Maxwell F.; Mazurenko, Anton; Chiu, Christie S.; Ji, Geoffrey; Greif, Daniel; Greiner, Markus

    2016-09-01

    Exotic phases of matter can emerge from strong correlations in quantum many-body systems. Quantum gas microscopy affords the opportunity to study these correlations with unprecedented detail. Here, we report site-resolved observations of antiferromagnetic correlations in a two-dimensional, Hubbard-regime optical lattice and demonstrate the ability to measure the spin-correlation function over any distance. We measure the in situ distributions of the particle density and magnetic correlations, extract thermodynamic quantities from comparisons to theory, and observe statistically significant correlations over three lattice sites. The temperatures that we reach approach the limits of available numerical simulations. The direct access to many-body physics at the single-particle level demonstrated by our results will further our understanding of how the interplay of motion and magnetism gives rise to new states of matter.

  4. Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase.

    PubMed

    Wilcox, S K; Putnam, C D; Sastry, M; Blankenship, J; Chazin, W J; McRee, D E; Goodin, D B

    1998-12-01

    The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

  5. Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles

    PubMed Central

    2016-01-01

    Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne–azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging. PMID:27652605

  6. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

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

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  8. Functional group placement in protein binding sites: a comparison of GRID and MCSS

    NASA Astrophysics Data System (ADS)

    Bitetti-Putzer, Ryan; Joseph-McCarthy, Diane; Hogle, James M.; Karplus, Martin

    2001-10-01

    One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

  9. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

    PubMed Central

    2010-01-01

    Background Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. Results We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. Conclusion Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene. PMID:20149245

  10. Excited state potential energy surfaces and their interactions in Fe(IV)=O active sites.

    PubMed

    Srnec, Martin; Wong, Shaun D; Solomon, Edward I

    2014-12-21

    The non-heme ferryl active sites are of significant interest for their application in biomedical and green catalysis. These sites have been shown to have an S = 1 or S = 2 ground spin state; the latter is functional in biology. Low-temperature magnetic circular dichroism (LT MCD) spectroscopy probes the nature of the excited states in these species including ligand-field (LF) states that are otherwise difficult to study by other spectroscopies. In particular, the temperature dependences of MCD features enable their unambiguous assignment and thus determination of the low-lying excited states in two prototypical S = 1 and S = 2 NHFe(IV)[double bond, length as m-dash]O complexes. Furthermore, some MCD bands exhibit vibronic structures that allow mapping of excited-state interactions and their effects on the potential energy surfaces (PESs). For the S = 2 species, there is also an unusual spectral feature in both near-infrared absorption and MCD spectra - Fano antiresonance (dip in Abs) and Fano resonance (sharp peak in MCD) that indicates the weak spin-orbit coupling of an S = 1 state with the S = 2 LF state. These experimental data are correlated with quantum-chemical calculations that are further extended to analyze the low-lying electronic states and the evolution of their multiconfigurational characters along the Fe-O PESs. These investigations show that the lowest-energy states develop oxyl Fe(III) character at distances that are relevant to the transition state (TS) for H-atom abstraction and define the frontier molecular orbitals that participate in the reactivity of S = 1 vs. S = 2 non-heme Fe(IV)[double bond, length as m-dash]O active sites. The S = 1 species has only one available channel that requires the C-H bond of a substrate to approach perpendicular to the Fe-oxo bond (the π channel). In contrast, there are three channels (one σ and two π) available for the S = 2 non-heme Fe(IV)[double bond, length as m-dash]O system allowing C-H substrate approach

  11. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  12. Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor*

    PubMed Central

    Bray, Lauriane; Froment, Carine; Pardo, Pierre; Candotto, Cédric; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine; Moulédous, Lionel

    2014-01-01

    The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, 412TNST415 at the end of the C terminus of the receptor, and additional sites involved in desensitization (372TS373) and internalization (Ser395). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns. PMID:25326382

  13. Effect of H2 binding on the nonadiabatic transition probability between singlet and triplet states of the [NiFe]-hydrogenase active site.

    PubMed

    Kaliakin, Danil S; Zaari, Ryan R; Varganov, Sergey A

    2015-02-12

    We investigate the effect of H2 binding on the spin-forbidden nonadiabatic transition probability between the lowest energy singlet and triplet electronic states of [NiFe]-hydrogenase active site model, using a velocity averaged Landau-Zener theory. Density functional and multireference perturbation theories were used to provide parameters for the Landau-Zener calculations. It was found that variation of the torsion angle between the terminal thiolate ligands around the Ni center induces an intersystem crossing between the lowest energy singlet and triplet electronic states in the bare active site and in the active site with bound H2. Potential energy curves between the singlet and triplet minima along the torsion angle and H2 binding energies to the two spin states were calculated. Upon H2 binding to the active site, there is a decrease in the torsion angle at the minimum energy crossing point between the singlet and triplet states. The probability of nonadiabatic transitions at temperatures between 270 and 370 K ranges from 35% to 32% for the active site with bound H2 and from 42% to 38% for the bare active site, thus indicating the importance of spin-forbidden nonadiabatic pathways for H2 binding on the [NiFe]-hydrogenase active site.

  14. Preliminary results of receiver function analyses at three sites across the Bransfield Strait, Antarctica

    NASA Astrophysics Data System (ADS)

    Martínez Arévalo, C.; Mancilla, F.; Almendros, J.; Aznarte, J. L.; Alguacil, G.

    2009-04-01

    In February 2008, in the framework of the International Polar Year 2008-2009 under grant POL2006-08663 of the spanish Ministry of Education, we deployed three broadband seismometers at points forming a N-S profile across the Bransfield Strait, Antarctica, in the region between the South Shetland Islands and the Antarctic Peninsula. This region is very interesting from a tectonic point of view, due to the opening of the rift of the Bransfield and the presence of the South Shetland microplate. For logistic reasons, our instruments where located in the vicinity of the Antarctic bases Juan Carlos I (Livingston Island), Gabriel de Castilla (Deception Island), and Primavera (Antarctic Peninsula). Each seismic station consisted of a broad-band, three-component electrolitic seismometer equiped with a 24-bit data acquisition system. The energy was provided by wind generators and solar panels connected to a battery bank, a combination that has been able to provide enough power at all sites, even during the Antarctic winter. All components were designed to function under the extreme conditions of the Antarctic weather. The main objective of this experiment was to use receiver function techniques on teleseism data to investigate the structure of the crust under the sites, in order to compare with other studies and shed light on the structure and tectonics of the region. During this past year, we have recorded several tens of teleseisms at distances appropriate for receiver function analyses. Preliminary results will add information on critical issues regarding the structure of the Bransfield Strait region. Although the Earth models obtained will contain extra information, we are specially interested in the determination of the depth of the Moho at each site, a controversial point in this area. These results will be compared with estimates obtained by different techniques. We are also interested in the determination of the vertical extent of the magma chamber recently imaged by

  15. Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

    PubMed Central

    Kharytonchyk, Siarhei; Monti, Sarah; Smaldino, Philip J.; Van, Verna; Bolden, Nicholas C.; Brown, Joshua D.; Russo, Emily; Swanson, Canessa; Shuey, Alex; Telesnitsky, Alice; Summers, Michael F.

    2016-01-01

    The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3–R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine (Cap1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines (Cap2G or Cap3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer–dimer equilibrium properties of Cap1G, Cap2G, and Cap3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap1G 5′-leader RNA adopts a dimeric structure, the Cap2G and Cap3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimer-promoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine. PMID:27834211

  16. On the role of the conformational flexibility of the active-site lid on the allosteric kinetics of glucosamine-6-phosphate deaminase.

    PubMed

    Bustos-Jaimes, Ismael; Sosa-Peinado, Alejandro; Rudiño-Piñera, Enrique; Horjales, Eduardo; Calcagno, Mario L

    2002-05-24

    The active site of glucosamine-6-phosphate deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) has a complex lid formed by two antiparallel beta-strands connected by a helix-loop segment (158-187). This motif contains Arg172, which is a residue involved in binding the substrate in the active-site, and three residues that are part of the allosteric site, Arg158, Lys160 and Thr161. This dual binding role of the motif forming the lid suggests that it plays a key role in the functional coupling between active and allosteric sites. Previous crystallographic work showed that the temperature coefficients of the active-site lid are very large when the enzyme is in its T allosteric state. These coefficients decrease in the R state, thus suggesting that this motif changes its conformational flexibility as a consequence of the allosteric transition. In order to explore the possible connection between the conformational flexibility of the lid and the function of the deaminase, we constructed the site-directed mutant Phe174-Ala. Phe174 is located at the C-end of the lid helix and its side-chain establishes hydrophobic interactions with the remainder of the enzyme. The crystallographic structure of the T state of Phe174-Ala deaminase, determined at 2.02 A resolution, shows no density for the segment 162-181, which is part of the active-site lid (PDB 1JT9). This mutant form of the enzyme is essentially inactive in the absence of the allosteric activator, N-acetylglucosamine-6-P although it recovers its activity up to the wild-type level in the presence of this ligand. Spectrometric and binding studies show that inactivity is due to the inability of the active-site to bind ligands when the allosteric site is empty. These data indicate that the conformational flexibility of the active-site lid critically alters the binding properties of the active site, and that the occupation of the allosteric site restores the lid conformational flexibility to a functional state.

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

    PubMed

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

    2016-08-01

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

  18. Hydrogen adsorption on functionalized nanoporous activated carbons.

    PubMed

    Zhao, X B; Xiao, B; Fletcher, A J; Thomas, K M

    2005-05-12

    There is considerable interest in hydrogen adsorption on carbon nanotubes and porous carbons as a method of storage for transport and related energy applications. This investigation has involved a systematic investigation of the role of functional groups and porous structure characteristics in determining the hydrogen adsorption characteristics of porous carbons. Suites of carbons were prepared with a wide range of nitrogen and oxygen contents and types of functional groups to investigate their effect on hydrogen adsorption. The porous structures of the carbons were characterized by nitrogen (77 K) and carbon dioxide (273 K) adsorption methods. Hydrogen adsorption isotherms were studied at 77 K and pressure up to 100 kPa. All the isotherms were Type I in the IUPAC classification scheme. Hydrogen isobars indicated that the adsorption of hydrogen is very temperature dependent with little or no hydrogen adsorption above 195 K. The isosteric enthalpies of adsorption at zero surface coverage were obtained using a virial equation, while the values at various surface coverages were obtained from the van't Hoff isochore. The values were in the range 3.9-5.2 kJ mol(-1) for the carbons studied. The thermodynamics of the adsorption process are discussed in relation to temperature limitations for hydrogen storage applications. The maximum amounts of hydrogen adsorbed correlated with the micropore volume obtained from extrapolation of the Dubinin-Radushkevich equation for carbon dioxide adsorption. Functional groups have a small detrimental effect on hydrogen adsorption, and this is related to decreased adsorbate-adsorbent and increased adsorbate-adsorbate interactions.

  19. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  20. A site-directed mutagenesis analysis of tNOX functional domains

    NASA Technical Reports Server (NTRS)

    Chueh, Pin-Ju; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

  1. Usage Of New Activation Function In Neuro-Symbolic Integration

    SciTech Connect

    Sathasivam, Saratha

    2010-12-23

    New activation function is examined for its ability to accelerate the performance of doing logic programming in Hopfield network. This method has a higher capacity and upgrades the neuro symbolic integration. Computer simulations are carried out to validate the effectiveness of the new activation function. Empirical results obtained support our theory.

  2. The hydrogen chemistry of the FeMo-co active site of nitrogenase.

    PubMed

    Dance, Ian

    2005-08-10

    The chemical mechanism by which nitrogenase enzymes catalyze the hydrogenation of N(2) (and other multiply bonded substrates) at the N(c)Fe(7)MoS(9)(homocitrate) active site (FeMo-co) is unknown, despite the accumulation of much data on enzyme reactivity and the influences of key amino acids surrounding FeMo-co. The mutual influences of H(2), substrates, and the inhibitor CO on reactivity are key experimental tests for postulated mechanisms. Fundamental to all aspects of mechanism is the accumulation of H atoms (from e(-) + H(+)) on FeMo-co, and the generation and influences of coordinated H(2). Here, I argue that the first introduction of H is via a water chain terminating at water 679 (PDB structure , Azotobacter vinelandii) to one of the mu(3)-S atoms (S3B) of FeMo-co. Next, using validated density functional calculations of a full chemical representation of FeMo-co and its connected residues (alpha-275(Cys), alpha-442(His)), I have characterized more than 80 possibilities for the coordination of up to three H atoms, and H(2), and H + H(2), on the S2A, Fe2, S2B, Fe6, S3B domain of FeMo-co, which is favored by recent targeted mutagenesis results. Included are calculated reaction profiles for movements of H atoms (between S and Fe, and between Fe and Fe), for the generation of Fe-H(2), for association and dissociation of Fe-H(2) at various reduction levels, and for H/H(2) exchange. This is new hydrogen chemistry on an unprecedented coordination frame, with some similarities to established hydrogen coordination chemistry, and with unexpected and unprecedented structures such as Fe(S)(3)(H(2))(2)(H) octahedral coordination. General principles for the hydrogen chemistry of FeMo-co include (1) the stereochemical mobility of H bound to mu(3)-S, (2) the differentiated endo- and exo- positions at Fe for coordination of H and/or H(2), and (3) coordinative allosteric influences in which structural and dynamic aspects of coordination at one Fe atom are affected by

  3. An Electromagnetic Interference Study of Potential Transmitter Sites for the HF Active Auroral Research Program (HAARP)

    DTIC Science & Technology

    1993-07-19

    heating . The measurements described in this report were conducted at a number of candidate HAARP transmitter sites in the vicinity of Fairbanks...employ the High Power Auroral Stimulation (HIPAS) RF heating facility [1], located in the Chena River valley area near Fairbanks. HAARP will be an...Potential Transmitter Sites for the HF Active Auroral Research Program ( HAARP ) JOSEP11 A. GOLDSTEIN EDWARD 1. KENNEDY ADRIAN S. ELEY 4 IMICHlAEL A. RuPAR C

  4. Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues.

    PubMed

    Kuhn, Isabelle; Kellenberger, Esther; Cakir-Kiefer, Céline; Muller-Steffner, Hélène; Schuber, Francis

    2014-07-01

    Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.

  5. Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

    PubMed

    Martinez, Salette; Wu, Rui; Krzywda, Karoline; Opalka, Veronika; Chan, Hei; Liu, Dali; Holz, Richard C

    2015-07-01

    A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1% of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active