Combining specificity determining and conserved residues improves functional site prediction

Directory of Open Access Journals (Sweden)

Gelfand Mikhail S

2009-06-01

Full Text Available Abstract Background Predicting the location of functionally important sites from protein sequence and/or structure is a long-standing problem in computational biology. Most current approaches make use of sequence conservation, assuming that amino acid residues conserved within a protein family are most likely to be functionally important. Most often these approaches do not consider many residues that act to define specific sub-functions within a family, or they make no distinction between residues important for function and those more relevant for maintaining structure (e.g. in the hydrophobic core. Many protein families bind and/or act on a variety of ligands, meaning that conserved residues often only bind a common ligand sub-structure or perform general catalytic activities. Results Here we present a novel method for functional site prediction based on identification of conserved positions, as well as those responsible for determining ligand specificity. We define Specificity-Determining Positions (SDPs, as those occupied by conserved residues within sub-groups of proteins in a family having a common specificity, but differ between groups, and are thus likely to account for specific recognition events. We benchmark the approach on enzyme families of known 3D structure with bound substrates, and find that in nearly all families residues predicted by SDPsite are in contact with the bound substrate, and that the addition of SDPs significantly improves functional site prediction accuracy. We apply SDPsite to various families of proteins containing known three-dimensional structures, but lacking clear functional annotations, and discusse several illustrative examples. Conclusion The results suggest a better means to predict functional details for the thousands of protein structures determined prior to a clear understanding of molecular function.

Analysis and radiological assessment of residues containing NORM materials resulting from earlier activities including modelling of typical industrial residues. Pt. 1. Historical investigation of the radiological relevance of NORM residues and concepts for site identification

International Nuclear Information System (INIS)

Reichelt, Andreas; Niedermayer, Matthias; Sitte, Beate; Hamel, Peter Michael

2007-01-01

Natural radionuclides are part of the human environment and of the raw materials used. Technical processes may cause their accumulation in residues, and the result will be so-called NORM materials (Naturally occurring radioactive material). The amended Radiation Protection Ordinance (StrlSchV 2001) specifies how the public should be protected, but there are also residues dating back before the issuing of the StrlSchV 2001, the so-called NORM residues. The project intended to assess the risks resulting from these residues. It comprises four parts. Part 1 was for clarification of the radiological relevance of NORM residues and for the development of concepts to detect them. The criterion for their radiological relevance was their activity per mass unit and the material volume accumulated through the centuries. The former was calculated from a wide bibliographic search in the relevant literature on radiation protection, while the mass volume was obtained by a detailed historical search of the consumption of materials that may leave NORM residues. These are, in particular, residues from coal and ore mining and processing. To identify concrete sites, relevant data sources were identified, and a concept for identification of concrete NORM residues was developed on this basis. (orig.) [de

Cys-X scanning for expansion of active-site residues and modulation of catalytic functions in a glutathione transferase.

Science.gov (United States)

Norrgård, Malena A; Hellman, Ulf; Mannervik, Bengt

2011-05-13

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

Cloning, Site-Directed Mutagenesis, and Functional Analysis of Active Residues in Lymantria dispar Chitinase.

Science.gov (United States)

Fan, Xiao-Jun; Yang, Chun; Zhang, Chang; Ren, Hui; Zhang, Jian-Dong

2018-01-01

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc) 3 . Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc) 3 , while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k cat and k cat / K m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.

REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCES HALIDE BINDING AND CATALYTIC ACTIVITY

NARCIS (Netherlands)

KENNES, C; PRIES, F; KROOSHOF, GH; BOKMA, E; Kingma, Jacob; JANSSEN, DB

1995-01-01

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues

Residual herbicide study on selected Hanford Site roadsides

Energy Technology Data Exchange (ETDEWEB)

Smith, J.L.; Kemp, C.J.; Sackschewsky, M.R.

1993-08-01

Westinghouse Hanford Company routinely treats roadsides with herbicides to control undesirable plant growth. An experiment was conducted to test perennial grass germination in soils adjacent to roadways of the Hanford Site. The primary variable was the distance from the roadside. A simple germination test was executed in a controlled-environment chamber to determine the residual effects of these applications. As expected, the greatest herbicide activity was found directly adjacent to the roadway, approximately 0 to 20 ft (0 to 6.3 m) from the roadway.

A tool for calculating binding-site residues on proteins from PDB structures

Directory of Open Access Journals (Sweden)

Hu Jing

2009-08-01

Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

Science.gov (United States)

Farb, Joshua N; Morrical, Scott W

2009-01-16

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

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

Energy Technology Data Exchange (ETDEWEB)

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.

Roles of the active site residues and metal cofactors in noncanonical base-pairing during catalysis by human DNA polymerase iota.

Science.gov (United States)

Makarova, Alena V; Ignatov, Artem; Miropolskaya, Nataliya; Kulbachinskiy, Andrey

2014-10-01

Human DNA polymerase iota (Pol ι) is a Y-family polymerase that can bypass various DNA lesions but possesses very low fidelity of DNA synthesis in vitro. Structural analysis of Pol ι revealed a narrow active site that promotes noncanonical base-pairing during catalysis. To better understand the structure-function relationships in the active site of Pol ι we investigated substitutions of individual amino acid residues in its fingers domain that contact either the templating or the incoming nucleotide. Two of the substitutions, Y39A and Q59A, significantly decreased the catalytic activity but improved the fidelity of Pol ι. Surprisingly, in the presence of Mn(2+) ions, the wild-type and mutant Pol ι variants efficiently incorporated nucleotides opposite template purines containing modifications that disrupted either Hoogsteen or Watson-Crick base-pairing, suggesting that Pol ι may use various types of interactions during nucleotide addition. In contrast, in Mg(2+) reactions, wild-type Pol ι was dependent on Hoogsteen base-pairing, the Y39A mutant was essentially inactive, and the Q59A mutant promoted Watson-Crick interactions with template purines. The results suggest that Pol ι utilizes distinct mechanisms of nucleotide incorporation depending on the metal cofactor and reveal important roles of specific residues from the fingers domain in base-pairing and catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Derivation of uranium residual radioactive material guidelines for the Ventron site

International Nuclear Information System (INIS)

Loureiro, C.; Yu, C.; Jones, L.

1992-03-01

Residual radioactive material guidelines for uranium were derived for the Ventron site in Beverly, Massachusetts. This site has been identified for remedial action under the Formerly Utilized Sites Remedial Action Program of the US Department of Energy (DOE). The derivations for the single radionuclides and the total uranium guidelines were based on the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the Ventron site should not exceed a dose of 100 mrem/yr following remedial action. The DOE residual radioactive material guideline computer code, RESRAD, which implements the methodology described in the DOE manual for implementing residual radioactive material guidelines, was used in this evaluation

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

Science.gov (United States)

Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan; Dougherty, Vi

2017-02-14

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in k cat /K m and k red for (S)-6-hydroxynicotine, respectively, with larger effects on the k cat /K m value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the k cat /K m value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the k cat /K m value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

Derivation of uranium residual radioactive material guidelines for the Shpack site

International Nuclear Information System (INIS)

Cheng, J.J.; Yu, C.; Monette, F.; Jones, L.

1991-08-01

Residual radioactive material guidelines for uranium were derived for the Shpack site in Norton, Massachusetts. This site has been identified for remedial action under the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the US Department of Energy (DOE). The uranium guidelines were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the Shpack site should not exceed a dose of 100 mrem/yr following decontamination. The DOE residual radioactive material guideline computer code, RESRAD, which implements the methodology described in the DOE manual for implementing residual radioactive material guidelines, was used in this evaluation. Three potential scenarios were considered for the site; the scenarios vary with regard to time spent at the site, sources of water used, and sources of food consumed. The results of the evaluation indicate that the basic dose limit of 100 mrem/yr will not be exceeded for uranium (including uranium-234, uranium-235, and uranium-238) within 1000 years, provided that the soil concentration of combined uranium (uranium-234 and uranium-238) at the Shpack site does not exceed the following levels: 2500 pCi/g for Scenario A (recreationist: the expected scenario); 1100 pCi/g for Scenario B (industrial worker: a plausible scenario); and 53 pCi/g for Scenario C (resident farmer using a well water as the only water source: a possible but unlikely scenario). The uranium guidelines derived in this report apply to the combined activity concentration of uranium-234 and uranium-238 and were calculated on the basis of a dose of 100 mrem/yr. In setting the actual uranium guidelines for the Shpack site, DOE will apply the as low as reasonably achievable (ALARA) policy to the decision-making process, along with other factors, such as whether a particular scenario is reasonable and appropriate. 8 refs., 2 figs., 8 tabs

A radiographic study on the prevalence of knife-edge residual alveolar ridge at proposed dental implant sites

International Nuclear Information System (INIS)

AlFaleh, Wafaa

2009-01-01

Dental implants are widely used in restoration of completely or partially edentulous dental arches. Before placement of endosseous implants in the jaws, both the quantity and quality of the residual ridge must be assessed radiographically. Remodeling activity after tooth extraction is localized primarily at the crestal area of the residual ridges, resulting in reduction of the height of bone and creation of various three-dimensional shapes of the residual ridges. When bone resorption at the lingual and buccal aspects is greater than that at the crestal area, a knife-edge type of residual ridge develops. The aim of this study was to evaluate the prevalence of the knife-edge morphology of the residual alveolar bone at proposed implant sites in partially or completely edentulous patients. Computed tomography (CT) cross-sectional images of the upper and lower jaws were assessed at the proposed sites before implant placement. Images of 258 proposed implant sites belonging to 30 patients were assessed radiographically. In 120 proposed implant sites out of 258 (46.5%), the residual alveolar ridge had a knife-edge configuration, the majority belonging to completely edentulous patients who lost their teeth more than ten years previously. High prevalence of knife-edge ridge was found, therefore, replacement of missing teeth by immediate implant is recommended to prevent atrophy or knife-edge morphology of the residual ridge. (author)

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

Energy Technology Data Exchange (ETDEWEB)

Tomkinson, B.; Wernstedt, C.; Hellman, U.; Zetterqvist, Oe.

1987-11-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.

Inventory of sites used to develop residual radioactivity criteria

International Nuclear Information System (INIS)

Ronca-Battista, M.; Hardeman, J.C. Jr.

1986-01-01

The US Environmental Protection Agency (EPA), in conjunction with the National Conference of Radiation Control Program Directors (CRCPD), has compiled an inventory of never licensed or otherwise poorly documented sites that may be contaminated with radioactive materials. This effort is in support of the EPA's development of radiation protection criteria for residual radioactivity at decommissioned sites. The inventory will help to establish the range of circumstances for which criteria are needed, as well as the suitability of candidate criteria for actual situations. The information will also be used to develop model sites and facilities for analyzing technical and economic feasibility of residual radioactivity criteria and to assess costs and benefits of alternate criteria. Relevant information about each site, such as radionuclides, waste forms, and quantities present will be included in the inventory when such information is available. The CRCPD has requested that each State radiation control agency furnish the information for the inventory. The inventory supplements the relatively extensive documentation of sites regulated by Federal or State agencies with information on old or unlicensed sites, such as old waste storage sites or radium ore processing facilities

Engineering assessment of radioactive sands and residues, Lowman Site, Lowman, Idaho

International Nuclear Information System (INIS)

1981-09-01

Ford, Bacon and Davis Utah Inc. has reevaluated the Lowman site in order to revise the December 1977 engineering assessment of the problems resulting from the existence of radioactive sands and residues at Lowman, Idaho. This engineering assessment has included the preparation of topographic maps, the performance of core drillings and radiometric measurements sufficient to determine areas and volumes of radioactive sands and residues and radiation exposure of individuals and nearby populations, and investigations of site hydrology and meteorology, and the evaluation and costing of alternative corrective actions. Radon gas released from the 191,000 tons of radioactive sands, residues, and contaminated soils at the Lowman site constitutes the most significant environmental impact, although windblown radioactive sands and external gamma radiation also are factors

Derivation of uranium residual radioactive material guidelines for the Elza Gate Site

International Nuclear Information System (INIS)

Cheng, J.J.; Yu, C.; Devgun, J.S.

1991-02-01

Residual radioactive material guidelines for uranium were derived for a large, homogeneously contaminated area at the Elza Gate Site in Oak Ridge, Tennessee. The derivation of the single-nuclide and total uranium guidelines was based on the requirement that the 50-year committed effective dose equivalent to hypothetical individual who lives or works in the immediate vicinity of the Elza Gate Site should not exceed a dose of 100 mrem/yr following decontamination. The DOE residual radioactive guideline computer code RESRAD was used in this evaluation. Four potential scenarios were considered for the site; the scenarios vary with regard to time spent at the site, sources of water used, and sources of food consumed. The results of the evaluation indicate that the basic dose limit of 100 mrem/yr will not be exceeded for uranium within 1000 years, provided that the soil concentration of uranium at the Elza Gate Site does not exceed the following levels: 1800 pCi/g for Scenario A (industrial worker: the expected scenario); 4000 pCi/g for Scenario B (recreationist: a plausible scenario); 470 pCi/g for Scenario C (resident farmer using pond water as the only water source: a possible but unlikely scenario); and 120 pCi/g for Scenario D (resident farmer using well water as the only water source: a possible but unlikely scenario). The uranium guideline applies to the total activity concentration of uranium isotopes in their natural activity concentration ratio of 1:1: 0.046. These guidelines are calculated on the basis of a dose of 100 mrem/yr. In setting the actual uranium guideline for the Elza Gate Site, the DOE will apply the as low as reasonably achievable (ALARA) policy to the decision-making process, along with other factors, such as determining whether a particular scenario is reasonable and appropriate. 10 refs., 3 figs., 7 tabs

Derivation of guidelines for uranium residual radioactive material in soil at the Colonie Site, Colonie, New York

International Nuclear Information System (INIS)

Dunning, D.

1996-05-01

Residual radioactive material guidelines for uranium in soil were derived for the Colonie site located in Colonie, New York. This site has been designated for remedial action under the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the U.S. Department of Energy (DOE). The site became contaminated with radioactive material as a result of operations conducted by National Lead (NL) Industries from 1958 to 1984; these activities included brass foundry operations, electroplating of metal products, machining of various components using depleted uranium, and limited work with small amounts of enriched uranium and thorium. The Colonie site comprises the former NL Industries property, now designated the Colonie Interim Storage Site (CISS), and 56 vicinity properties contaminated by fallout from airborne emissions; 53 of the vicinity properties were previously remediated between 1984 and 1988. In 1984, DOE accepted ownership of the CISS property from NL Industries. Residual radioactive material guidelines for individual radionuclides and total uranium were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the site should not exceed a dose of 30 mrem/yr following remedial action for the current use and likely future use scenarios or a dose of 100 mrem/yr for less likely future use scenarios. The DOE residual radioactive material guideline computer code, RESRAD, was used in this evaluation; RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines

The dapE-encoded N-succinyl-L,L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

Science.gov (United States)

Gillner, Danuta M.; Bienvenue, David L.; Nocek, Boguslaw P.; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C.

2009-01-01

The catalytic and structural properties of the H67A and H349A altered dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from H. influenzae were investigated. Based on sequence alignment with CPG2 both H67 and H349 were predicted to be Zn(II) ligands. Catalytic activity was observed for the H67A altered DapE enzyme which exhibited kcat = 1.5 ± 0.5 sec−1 and Km = 1.4 ± 0.3 mM. No catalytic activity was observed for H349A under the experimental conditions used. The EPR and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to WT DapE after the addition of a single Co(II) ion. The addition of one equivalent of Co(II) to H67A altered DapE provides spectra that are very different from the first Co(II) binding site of the WT enzyme, but similar to the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from N. meningitidis as a template and superimposed on the structure of AAP. This homology structure confirms the assignment of H67 and H349 as active site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP, all of the amino acid residues of DapE are nearly identical. PMID:18712420

Mutational properties of amino acid residues: implications for evolvability of phosphorylatable residues

DEFF Research Database (Denmark)

Creixell, Pau; Schoof, Erwin M.; Tan, Chris Soon Heng

2012-01-01

in terms of their mutational activity. Moreover, we highlight the importance of the genetic code and physico-chemical properties of the amino acid residues as likely causes of these inequalities and uncover serine as a mutational hot spot. Finally, we explore the consequences that these different......; it is typically assumed that all amino acid residues are equally likely to mutate or to result from a mutation. Here, by reconstructing ancestral sequences and computing mutational probabilities for all the amino acid residues, we refute this assumption and show extensive inequalities between different residues...... mutational properties have on phosphorylation site evolution, showing that a higher degree of evolvability exists for phosphorylated threonine and, to a lesser extent, serine in comparison with tyrosine residues. As exemplified by the suppression of serine's mutational activity in phosphorylation sites, our...

Identification of key residues for pH dependent activation of violaxanthin de-epoxidase from Arabidopsis thaliana.

Science.gov (United States)

Fufezan, Christian; Simionato, Diana; Morosinotto, Tomas

2012-01-01

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pK(a) values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121-Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role.

Identification of key residues for pH dependent activation of violaxanthin de-epoxidase from Arabidopsis thaliana.

Directory of Open Access Journals (Sweden)

Christian Fufezan

Full Text Available Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pK(a values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206 caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121-Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role.

The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.

Science.gov (United States)

Gillner, Danuta M; Bienvenue, David L; Nocek, Boguslaw P; Joachimiak, Andrzej; Zachary, Vincentos; Bennett, Brian; Holz, Richard C

2009-01-01

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

Derivation of residual radioactive material guidelines for uranium in soil at the Middlesex Sampling Plant Site, Middlesex, New Jersey

International Nuclear Information System (INIS)

Dunning, D.E.

1995-02-01

Residual radioactive material guidelines for uranium in soil were derived for the Middlesex Sampling Plant (MSP) site in Middlesex, New Jersey. This site has been designated for remedial action under the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the US Department of Energy. The site became contaminated from operations conducted in support of the Manhattan Engineer District (MED) and the Atomic Energy Commission (AEC) between 1943 and 1967. Activities conducted at the site included sampling, storage, and shipment of uranium, thorium, and beryllium ores and residues. Uranium guidelines for single radioisotopes and total uranium were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual living or working in the immediate vicinity of the MSP site should not exceed a dose of 30 mrem/yr following remedial action for the current-use and likely future-use scenarios or a dose of 100 mrem/yr for less likely future-use scenarios. The RESRAD computer code, which implements the methodology described in the DOE manual for establishing residual radioactive material guidelines, was used in this evaluation. Four scenarios were considered for the site. These scenarios vary regarding future land use at the site, sources of water used, and sources of food consumed

Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

International Nuclear Information System (INIS)

Vlahos, C.J.

1987-01-01

The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with [ 14 C]pyruvate in the presence of NaCNBH 3 results in the incorporation of one mol of 14 C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with [3- 14 C]bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved

Methodology for determining acceptable residual radioactive contamination levels at decommissioned nuclear facilities/sites

International Nuclear Information System (INIS)

Watson, E.C.; Kennedy, W.E. Jr.; Hoenes, G.R.; Waite, D.A.

1979-01-01

The ultimate disposition of decommissioned nuclear facilities and their surrrounding sites depends upon the degree and type of residual contamination. Examination of existing guidelines and regulations has led to the conclusion that there is a need for a general method to derive residual radioactive contamination levels that are acceptable for public use of any decommissioned nuclear facility or site. This paper describes a methodology for determining acceptable residual radioactive contamination levels based on the concept of limiting the annual dose to members of the public. It is not the purpose of this paper to recommend or even propose dose limits for the exposure of the public to residual radioactive contamination left at decommissioned nuclear facilities or sites. Unrestricted release of facilities and/or land is based on the premise that the potential annual dose to any member of the public using this property from all possible exposure pathways will not exceed appropriate limits as may be defined by Federal regulatory agencies. For decommissioned land areas, consideration should be given to people living directly on previously contaminated areas, growing crops, grazing food animals and using well water. Mixtures of radionuclides in the residual contamination representative of fuel reprocessing plants, light water reactors and their respective sites are presented. These mixtures are then used to demonstrate the methodology. Example acceptable residual radioactive contamination levels, based on an assumed maximum annual dose of one millirem, are calculated for several selected times following shutdown of a facility. It is concluded that the methodology presented in this paper results in defensible acceptable residual contamination levels that are directly relatable to risk assessment with the proviso that an acceptable limit to the maximum annual dose will be established. (author)

Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

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Misquitta Stephanie A

2004-02-01

Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

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

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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.

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

DEFF Research Database (Denmark)

Madec, Edwige; Stensballe, Allan; Kjellström, Sven

2003-01-01

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing...... the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high...... mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant...

Summary of the engineering assessment of radioactive sands and residues, Lowman Site, Lowman, Idaho

International Nuclear Information System (INIS)

1981-09-01

Ford, Bacon and Davis Utah Inc. has reevaluated the Lowman site in order to revise the December 1977 engineering assessment of the problems resulting from the existence of radioactive sands and residues at Lowman, Idaho. This engineering assessment has included the preparation of topographic maps, the performance of core drillings and radiometric measurements sufficient to determine areas and volumes of radioactive sands and residues and radiation exposures of individuals and nearby populations, the investigations of site hydrology and meteorology, and the evaluation and costing of alternative corrective actions. Radon gas released from the 191,000 tons of radioactive sands, residues, and contaminated soils at the Lowman site constitutes the most significant environmental impact, although windblown radioactive sands and external gamma radiation also are factors

Mapping the active site of vaccinia virus RNA triphosphatase

International Nuclear Information System (INIS)

Gong Chunling; Shuman, Stewart

2003-01-01

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

Status policy and criteria for the remediation of sites with radioactive residues in China

International Nuclear Information System (INIS)

Yamin, Z.

1999-01-01

This paper introduces the status policy and criteria for remediation of sites with radioactive residues in China. We deal with the sites in difference patterns according to their circumstances. For the sites related with the decommissioning of the nuclear fuel cycle facilities, the programs for environmental restoration must be reviewed and approved by the State Environmental Protection Administration (SEPA). And the radioactive waste resulting from these facilities should be collected and eventually disposed of at the regional disposal repositories built by the country. For the sites related with use of radionuclides in medicine, research and industry, as well as the small scale extraction and processing of materials containing natural radionuclides, the provincial environmental authority is responsible for approving the restoration projects. These radioactive wastes should finally be sent to the special radioactive waste repositories built by the provincial environmental authorities. So far 21 waste repositories have been built in some provinces. More then 10 sites with radioactive residues have been restored. The standards for general public dose limit and soil residual radionuclides content have been established. (author)

Influence of amino acid residues near the active site of cytochrome P450 from Bacillus megaterium on the selectivity of n-octane oxidation to octanol regioisomers

Science.gov (United States)

Miyaji, Akimitsu; Baba, Toshihide

2017-09-01

A mutant of cytochrome P450 from Bacillus megaterium (CYP450BM-3) was prepared by replacing two alanine residues around active site of the enzyme, alanine 328 and alanine 82, with leucine and tryptophan, respectively. The CYP450BM-3 mutant produced 2-octanol selectively from n-octane under atmospheric temperature and pressure; its selectivity was 74%. Furthermore, the mutant produced 1-octanol, which is not produced by wild-type enzyme.

Derivation of residual radioactive material guidelines for the Laboratory for Energy-Related Health Research site

International Nuclear Information System (INIS)

Chapman, T.E.

1993-11-01

Residual radioactive material guidelines were derived for the Laboratory for Energy-Related Health Research (LEHR) Environmental Restoration (ER) site in Davis, California. The guideline derivation was based on a dose limit of 100 mrem/yr. The US Department of Energy (DOE) residual radioactive material guideline computer code was used in this evaluation. This code implements the methodology described in the DOE manual for implementing residual radioactive material guidelines. Three potential site utilization scenarios were considered with the assumption that following ER action, the site will be used without radiological restrictions. The defined scenarios vary with regard to use of the site, time spent at the site, and sources of food consumed. The results of the evaluation indicate that the basic dose limit of 100 mrem/yr will not be exceeded, provided that the soil concentrations of these radionuclides at the LEHR site do not exceed the scenario-specific values calculated by this study. Except for the extent of the contaminated zone (which is very conservative), assumptions used are as site-specific as possible, given available information. The derived guidelines are single- radionuclide guidelines and are linearly proportional to the dose limit used in the calculations. In setting the actual residual soil contamination guides for the LEHR site, DOE will apply the as low as reasonably achievable policy to the decision-making process, along with other factors such as whether a particular scenario is reasonable and appropriate, as well as using site-specific inputs to computer models based on data not yet fully determined

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

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

2007-06-01

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

Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum.

Science.gov (United States)

Qin, Ning; Shen, Yanbing; Yang, Xu; Su, Liqiu; Tang, Rui; Li, Wei; Wang, Min

2017-07-01

3-Ketosteroid-Δ 1 -dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.

Magnesium-adenosine diphosphate binding sites in wild-type creatine kinase and in mutants: role of aromatic residues probed by Raman and infrared spectroscopies.

Science.gov (United States)

Hagemann, H; Marcillat, O; Buchet, R; Vial, C

2000-08-08

Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO(2))]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes.

Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

Directory of Open Access Journals (Sweden)

Silvia Schumann

Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

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In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

Residual radioactivity in the soil of the Semipalatinsk Nuclear Test Site in the former USSR.

Science.gov (United States)

Yamamoto, M; Tsukatani, T; Katayama, Y

1996-08-01

This paper deals with our efforts to survey residual radioactivity in the soil sampled at the Semipalatinsk Nuclear Test Site and at off-site areas in Kazakhstan. The soil was sampled at the hypocenter where the first Soviet nuclear explosion was carried out on 29 August 1949, and at the bank of the crater called "Bolapan," which was formed by an underground nuclear detonation on 15 January 1965 along the Shagan River. As a comparison, other soil was also sampled in the cities of Kurchatov and Almaty. These data have allowed a preliminary evaluation of the contemporary radioactive contamination of the land in and around the test site. At the first nuclear explosion site and at Bolapan, higher than background levels of 239,240Pu with weapons-grade plutonium were detected together with fission and activation products such as 137Cs, 60Co, 152Eu, and 154Eu.

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity.

Science.gov (United States)

Li, Jing-Ya; Cui, Yong-Mei; Chen, Ling-Ling; Gu, Min; Li, Jia; Nan, Fa-Jun; Ye, Qi-Zhuang

2004-05-14

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

Science.gov (United States)

Desideri, A; Falconi, M; Polticelli, F; Bolognesi, M; Djinovic, K; Rotilio, G

1992-01-05

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

Directory of Open Access Journals (Sweden)

Md Zahid Kamal

Full Text Available Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

Science.gov (United States)

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

Assessment of residual DDE at four remediated Hanford waste sites, Richland, Washington

International Nuclear Information System (INIS)

Linville, J.K.

1999-01-01

The objectives of this study were to determine the extent and distribution of residual DDE, a metabolite of dichlorodiphenyltrichloroethane (DDT), across the four waste sites by sampling ground-dwelling insects and bird eggs, evaluating the use of insects for monitoring contamination pathways, and determining the species of passerine birds present and the number of nesting pairs utilizing the waste sites

Derivation of uranium residual radioactive material guidelines for the former Alba Craft Laboratory site, Oxford, Ohio

International Nuclear Information System (INIS)

Nimmagadda, M.; Faillace, E.; Yu, C.

1994-01-01

Residual radioactive material guidelines for uranium were derived for the former Alba Craft Laboratory site in Oxford, Ohio. This site has been identified for remedial action under the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the US Department of Energy (DOE). Single nuclide and total uranium guidelines were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the former Alba Craft Laboratory site should not exceed a dose of 30 mrem/yr following remedial action for the current use and likely future use scenarios or a dose of 100 mrem/yr for less likely future use scenarios (Yu et al. 1993). The DOE residual radioactive material guideline computer code, RESRAD, which implements the methodology described in the DOE manual for implementing residual radioactive material guidelines, was used in this evaluation

Some problems of residual activity measurements

International Nuclear Information System (INIS)

Katrik, P.; Mustafin, E.; Strasik, I.; Pavlovic, M.

2013-01-01

As a preparatory work for constructing the Facility for Antiproton and Ion Research (FAIR) at GSI Darmstadt, samples of copper were irradiated by 500 MeV/u 238 U ion beam and investigated by gamma-ray spectroscopy. The nuclides that contribute dominantly to the residual activity have been identified and their contributions have been quantified by two different methods: from the whole-target gamma spectra and by integration of depth-profiles of residual activity of individual nuclides. Results obtained by these two methods are compared and discussed in this paper. (authors)

Residual radioactivity in the soil of the Semipalatinsk Nuclear Test Site in the former USSR

International Nuclear Information System (INIS)

Yamamoto, Masayoshi; Tsukatani, Tsuneo; Katayama, Yukio

1996-01-01

This paper deals with our efforts to survey residual readioactivity in the soil sampled at teh Semipalatinsk Nuclear Test Site and at off-site areas in Kazakhstan. The soil sampled at the hypocenter where the first Soviet nuclear explosion was carried out on 29 August 1949, and at the bank of the crater called open-quotes Bolapan,close quotes which was formed by an underground nuclear detonation on 15 January 1965 along the Shagan River. As a comparison, other soil was also sampled in the cities of Kurchatov and Almaty. These data have allowed a preliminary evaluation of the contemporary radioactive contamination of the land in and around the test site. At the first nuclear explosion site and at Bolapan, higher than background levels of 239,240 Pu with weapons-grade plutonium were detected together with fission and activation products such as 137 Cs, 60 Co, 152 Eu, and 154 Eu. 20 refs., 3 figs., 5 tabs

An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

Directory of Open Access Journals (Sweden)

Ryan W Benson

Full Text Available DinB (DNA Pol IV is a translesion (TLS DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ, a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS. Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

Effect of soil-bound residues of malathion on microbial activities

International Nuclear Information System (INIS)

Hussain, A.; Iqbal, Z.; Asi, M.R.; Tahira, R.; Chudhary, J.A.

2001-01-01

The effect of soil-bound residues of malathion on CO/sub 2/ evolution, dehydrogenase activity and some nitrogen transformations in a loam soil was investigated under laboratory conditions. The soil samples containing bound residues arising from 10 mg g-1 of the applied malathion were mixed in equal quantity with fresh soil and compared with solvent extracted control soil without bound residues (extracted in the same way as soil containing bound residues). Another control comprising un extracted fresh soil without bound residues was also kept to study the effect of solvent extraction on the biological activity. Rate of Carbon mineralization (CO/sub 2/ evolution) was decreased in the presence of soil-bound residues of malathion. Bound residues also affected dehydrogenase activity of soil. Over 40% inhibition of dehydrogenase activity was observed after 4 days and the inhibition persisted at least for 12 days. Nitrogen mineralization was stimulated in soil containing bound residues of malathion and this stimulatory effect increased with time of incubation. Nitrification was partially inhibited in the presence of soil-bound residues of malathion. The inhibitory effect of the soil-bound residues on nitrification did not show much variation with time. The soil-bound residues did not affect denitrification rate (N/sub 2/O evolution). Nitrogen fixation (acetylene reduction) was partially inhibited in soil amended with bound residues of malathion and the inhibitory effect persisted for at least one week. In general, soil bound residues of malathion inhibited CO/sub 2/ evolution, dehydrogenase activity, nitrification and nitrogen fixation while mineralization of nitrogen was stimulated. Denitrification was not affected by the applied insecticide. (author)

Substrate binding in the active site of cytochrome P450cam

NARCIS (Netherlands)

Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

2005-01-01

We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

International Nuclear Information System (INIS)

Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F.; Kenny, J.W.; Thompson, G.A.

1990-01-01

The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB 3 H 4 or Ado[ 14 C]Met. Peptide sequencing of both 3 H- and 14 C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3 H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14 C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [ 14 C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

Development of METAL-ACTIVE SITE and ZINCCLUSTER tool to predict active site pockets.

Science.gov (United States)

Ajitha, M; Sundar, K; Arul Mugilan, S; Arumugam, S

2018-03-01

The advent of whole genome sequencing leads to increasing number of proteins with known amino acid sequences. Despite many efforts, the number of proteins with resolved three dimensional structures is still low. One of the challenging tasks the structural biologists face is the prediction of the interaction of metal ion with any protein for which the structure is unknown. Based on the information available in Protein Data Bank, a site (METALACTIVE INTERACTION) has been generated which displays information for significant high preferential and low-preferential combination of endogenous ligands for 49 metal ions. User can also gain information about the residues present in the first and second coordination sphere as it plays a major role in maintaining the structure and function of metalloproteins in biological system. In this paper, a novel computational tool (ZINCCLUSTER) is developed, which can predict the zinc metal binding sites of proteins even if only the primary sequence is known. The purpose of this tool is to predict the active site cluster of an uncharacterized protein based on its primary sequence or a 3D structure. The tool can predict amino acids interacting with a metal or vice versa. This tool is based on the occurrence of significant triplets and it is tested to have higher prediction accuracy when compared to that of other available techniques. © 2017 Wiley Periodicals, Inc.

Derivation of guidelines for uranium residual radioactive material in soil at the New Brunswick Site, Middlesex County, New Jersey

International Nuclear Information System (INIS)

Dunning, D.; Kamboj, S.; Nimmagadda, M.; Yu, C.

1996-02-01

Residual radioactive material guidelines for uranium in soil were derived for the New Brunswick Site, located in Middlesex County, New Jersey. This site has been designated for remedial action under the Formerly Utilized Sites Remedial Action Program of the US Department of Energy (DOE). Residual radioactive material guidelines for individual radionuclides of concern and total uranium were derived on the basis of the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the New Brunswick Site should not exceed a dose of 30 mrem/yr following remedial action for the current-use and likely future-use scenarios or a dose of 100 mrem/yr for less likely future-use scenarios. The DOE residual radioactive material guideline computer code, RESRAD, was used in this evaluation; RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines. The guidelines derived in this report are intended to apply to the remediation of these remaining residual radioactive materials at the site. The primary radionuclides of concern in these remaining materials are expected to be radium-226 and, to a lesser extent, natural uranium and thorium. The DOE has established generic cleanup guidelines for radium and thorium in soil; however, cleanup guidelines for other radionuclides must be derived on a site-specific basis

Phase II, Title I engineering assessment of radioactive sands and residues, Lowman Site, Lowman Idaho

Energy Technology Data Exchange (ETDEWEB)

1977-12-01

An engineering assessment was performed of the problems resulting from the existence of radioactive uranium sand residues at the Lowman, Idaho, site. Services normally include the preparation of topographic maps, the performance of core drillings and radiometric measurements sufficient to determine areas and volumes of tailings and other radium-contaminated materials, the evaluation of resulting investigations of site hydrology and meteorology, and the evaluation and costing of alternative corrective actions. Radon gas release from the 90,000 tons of sand residues at the Lowman site constitutes the most significant environmental impact, although external gamma radiation is also a factor. The two alternative actions presented are dike construction, fencing, and maintenance (Option I); and consolidation of the piles, addition of a 2-ft-thick stabilization cover, and on-site cleanup (Option II). Both options include remedial action at off-site structures. Cost estimates for the two options are $393,000 and $590,000.

Phase II, Title I engineering assessment of radioactive sands and residues, Lowman Site, Lowman Idaho

International Nuclear Information System (INIS)

1977-12-01

An engineering assessment was performed of the problems resulting from the existence of radioactive uranium sand residues at the Lowman, Idaho, site. Services normally include the preparation of topographic maps, the performance of core drillings and radiometric measurements sufficient to determine areas and volumes of tailings and other radium-contaminated materials, the evaluation of resulting investigations of site hydrology and meteorology, and the evaluation and costing of alternative corrective actions. Radon gas release from the 90,000 tons of sand residues at the Lowman site constitutes the most significant environmental impact, although external gamma radiation is also a factor. The two alternative actions presented are dike construction, fencing, and maintenance (Option I); and consolidation of the piles, addition of a 2-ft-thick stabilization cover, and on-site cleanup (Option II). Both options include remedial action at off-site structures. Cost estimates for the two options are $393,000 and $590,000

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

Science.gov (United States)

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

2008-09-01

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

Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis

NARCIS (Netherlands)

Bokma, Evert; Rozeboom, Henriëtte J.; Sibbald, Mark; Dijkstra, Bauke W.; Beintema, Jaap J.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the -1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

Science.gov (United States)

Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

2014-01-01

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

Single residue mutation in active site of serine acetyltransferase isoform 3 from Entamoeba histolytica assists in partial regaining of feedback inhibition by cysteine.

Directory of Open Access Journals (Sweden)

Sudhir Kumar

Full Text Available The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT and O-acetylserine sulfhydrylase (OASS are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K(m, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3 shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.

Strategy utilized for assessing baseline risks to human health from K-65 and metal oxide residues stored at the Fernald Site

International Nuclear Information System (INIS)

Harmon, J.E.; Janke, R.C.

1995-01-01

The U.S. Department of Energy (DOE) is responsible for cleanup activities at the Fernald Environmental Management Project (FEMP) site in southwestern Ohio. The 425-hectare site consists of a former 55-hectare Production Area, an adjacent Waste Storage Area and various support facilities. From 1952 until 1989, the FEMP processed uranium into metallic open-quotes feedclose quotes materials for other DOE facilities in the nation's defense program. In accordance with the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), the FEMP site is currently listed on the National Priorities List (NPL). To facilitate an expeditious cleanup effort, environmental issues associated with site cleanup are being managed under five operable units. This paper summarizes the risk assessment strategy employed to determine baseline human health risks associated with K-65 and metal oxide residues currently stored in Operable Unit 4. The K-65 and metal oxide residues were generated during the 1950s as a result of the extraction of uranium from uranium-bearing ores and concentrates. These residues are currently stored within Operable Unit 4 in concrete silos. Silos I and 2 contain approximately 6,120 cubic meters [m 3 ] (8,005 cubic yards [yd 3 ]) of K-65 residues, while silos 3 contains approximately 3890 m 3 (5,080 yd 3 ) of cold metal oxides. These concrete silos are beyond their design life and require remedial action. The risk assessment conducted for Operable Unit 4 constitutes the first detailed human health risk assessment to be approved by the Environmental Protection Agency (EPA) for the CERCLA clean-up effort at the FEMP Site. This paper discusses the FEMP's use of a Risk Information Quality Objective process in concert with the traditional risk assessment approach to determine baseline risk to human health and the environment posed by Operable Unit 4. A summary of the baseline risks to human health is also presented

Engineering evaluation of alternatives for the disposition of Niagara Falls Storage Site, its residues and wastes

International Nuclear Information System (INIS)

1984-01-01

The final disposition scenarios selected by DOE for assessment in this document are consistent with those stated in the Notice of Intent to prepare an Environmental Impact Statement (EIS) for the Niagara Falls Storage Site (NFSS) (DOE, 1983d) and the modifications to the alternatives resulting from the public scoping process. The scenarios are: take no action beyond interim remedial measures other than maintenance and surveillance of the NFSS; retain and manage the NFSS as a long-term waste management facility for the wastes and residues on the site; decontaminate, certify, and release the NFSS for other use, with long-term management of the wastes and residues at other DOE sites; and partially decontaminate the NFSS by removal and transport off site of only the more radioactive residues, and upgrade containment of the remaining wastes and residues on site. The objective of this document is to present to DOE the conceptual engineering, occupational radiation exposure, construction schedule, maintenance and surveillance requirements, and cost information relevant to design and implementation of each of the four scenarios. The specific alternatives within each scenario used as the basis for discussion in this document were evaluated on the bases of engineering considerations, technical feasibility, and regulatory requirements. Selected alternatives determined to be acceptable for each of the four final disposition scenarios for the NFSS were approved by DOE to be assessed and costed in this document. These alternatives are also the subject of the EIS for the NFSS currently being prepared by Argonne National Laboratory (ANL). 40 figures, 38 tables

Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

Science.gov (United States)

da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

2014-01-01

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

On-site treatment and landfilling of MSWI air pollution control residues

DEFF Research Database (Denmark)

Lundtorp, Kasper; Jensen, Dorthe Lærke; Sørensen, Mette Abildgaard

2003-01-01

the process, collected through the drainage system, contained large concentrations of salts (Cl: 14–30 g/l, Na: 4–9 g/l, K: 5–11 g/l, Ca: 2–12 g/l) but low concentrations of trace metals (e.g. Pb: 14–100 μg/l, Cd: leaching......Air pollution control (APC) residues from municipal solid waste incineration (MSWI) are difficult to landfill due to substantial leaching of trace metals. An on-site pretreatment prior to landfilling of APC-residues was investigated in terms of bench-scale experiments with a semidry APC...... of the leaching, concentrations of trace metals were reduced by up to four orders of magnitude....

Work plan for the remedial investigation/feasibility study-environmental assessment for the quarry residuals operable unit at the Weldon Spring Site

Energy Technology Data Exchange (ETDEWEB)

1994-01-01

The US Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, which is located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis. The Weldon Spring site consists of two noncontiguous areas -- the chemical plant area, which includes four raffinate pits, and the quarry. Cleanup activities at the Weldon Spring site are conducted in accordance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, incorporating the values of the National Environmental Policy Act (NEPA). The contents of the documents prepared for the project are not intended to represent a statement regarding the legal applicability of NEPA to remedial actions conducted under CERCLA. In accordance with the integrated CERCLA/NEPA approach, a remedial investigation/feasibility study-environmental assessment (RI/FS-EA) is being conducted to evaluate conditions and potential responses for the quarry residuals operable unit (QROU). This operable unit consists of the following areas and/or media: the residual material remaining at the Weldon Spring quarry after removal of the pond water and bulk waste; underlying groundwater; and other media located in the surrounding vicinity of the quarry, including adjacent soil, surface water, and sediment in Femme Osage Slough. This work plan identifies the activities within the RI/FS-EA process that are being proposed to address contamination remaining at the quarry area.

Work plan for the remedial investigation/feasibility study-environmental assessment for the quarry residuals operable unit at the Weldon Spring Site

International Nuclear Information System (INIS)

1994-01-01

The US Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, which is located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis. The Weldon Spring site consists of two noncontiguous areas -- the chemical plant area, which includes four raffinate pits, and the quarry. Cleanup activities at the Weldon Spring site are conducted in accordance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, incorporating the values of the National Environmental Policy Act (NEPA). The contents of the documents prepared for the project are not intended to represent a statement regarding the legal applicability of NEPA to remedial actions conducted under CERCLA. In accordance with the integrated CERCLA/NEPA approach, a remedial investigation/feasibility study-environmental assessment (RI/FS-EA) is being conducted to evaluate conditions and potential responses for the quarry residuals operable unit (QROU). This operable unit consists of the following areas and/or media: the residual material remaining at the Weldon Spring quarry after removal of the pond water and bulk waste; underlying groundwater; and other media located in the surrounding vicinity of the quarry, including adjacent soil, surface water, and sediment in Femme Osage Slough. This work plan identifies the activities within the RI/FS-EA process that are being proposed to address contamination remaining at the quarry area

Study on indium leaching from mechanically activated hard zinc residue

Directory of Open Access Journals (Sweden)

Yao J.H.

2011-01-01

Full Text Available In this study, changes in physicochemical properties and leachability of indium from mechanically activated hard zinc residue by planetary mill were investigated. The results showed that mechanical activation increased specific surface area, reaction activity of hard zinc residue, and decreased its particle size, which had a positive effect on indium extraction from hard zinc residue in hydrochloric acid solution. Kinetics of indium leaching from unmilled and activated hard zinc residue were also investigated, respectively. It was found that temperature had an obvious effect on indium leaching rate. Two different kinetic models corresponding to reactions which are diffusion controlled, [1-(1- x1/3]2=kt and (1-2x/3-(1-x2/3=kt were used to describe the kinetics of indium leaching from unmilled sample and activated sample, respectively. Their activation energies were determined to be 17.89 kJ/mol (umilled and 11.65 kJ/mol (activated within the temperature range of 30°C to 90°C, which is characteristic for a diffusion controlled process. The values of activation energy demonstrated that the leaching reaction of indium became less sensitive to temperature after hard zinc residue mechanically activated by planetary mill.

Chemical activation of gasification carbon residue for phosphate removal

Science.gov (United States)

Kilpimaa, Sari; Runtti, Hanna; Lassi, Ulla; Kuokkanen, Toivo

2012-05-01

Recycling of waste materials provides an economical and environmentally significant method to reduce the amount of waste. Bioash formed in the gasification process possesses a notable amount of unburned carbon and therefore it can be called a carbon residue. After chemical activation carbon residue could be use to replace activated carbon for example in wastewater purification processes. The effect of chemical activation process variables such as chemical agents and contact time in the chemical activation process were investigated. This study also explored the effectiveness of the chemically activated carbon residue for the removal of phosphate from an aqueous solution. The experimental adsorption study was performed in a batch reactor and the influence of adsorption time, initial phosphate concentration and pH was studied. Due to the carbon residue's low cost and high adsorption capacity, this type of waste has the potential to be utilised for the cost-effective removal of phosphate from wastewaters. Potential adsorbents could be prepared from these carbonaceous by-products and used as an adsorbent for phosphate removal.

Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop

Directory of Open Access Journals (Sweden)

Christian B. Oehlenschlæger

2017-11-01

Full Text Available PSY1R is a leucine-rich repeat (LRR receptor-like kinase (RLK previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1 and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Energy Technology Data Exchange (ETDEWEB)

Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. (Friedrich Miescher-Institut, Basel (Switzerland))

1991-02-26

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase.

Science.gov (United States)

Fecker, Tobias; Galaz-Davison, Pablo; Engelberger, Felipe; Narui, Yoshie; Sotomayor, Marcos; Parra, Loreto P; Ramírez-Sarmiento, César A

2018-03-27

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic

Active site mutations change the cleavage specificity of neprilysin.

Directory of Open Access Journals (Sweden)

Travis Sexton

Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

Science.gov (United States)

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

Science.gov (United States)

Frederick, Thomas E; Peng, Jeffrey W

2018-01-01

Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

Old radioactive waste storage sites

International Nuclear Information System (INIS)

2008-01-01

After a recall of the regulatory context for the management of old sites used for the storage of radioactive wastes with respect with their activity, the concerned products, the disposal or storage type, this document describes AREVA's involvement in the radioactive waste management process in France. Then, for the different kinds of sites (currently operated sites having radioactive waste storage, storage sites for uranium mineral processing residues), it indicates their location and name, their regulatory status and their control authority, the reference documents. It briefly presents the investigation on the long term impact of uranium mineral processing residues on health and environment, evokes some aspects of public information transparency, and presents the activities of an expertise group on old uranium mines. The examples of the sites of Bellezane (uranium mineral processing residues) and COMURHEX Malvesi (assessment of underground and surface water quality at the vicinity of this installation) are given in appendix

Proposed plan for remedial action at the quarry residuals operable unit of the Weldon Spring Site

International Nuclear Information System (INIS)

1998-03-01

This proposed plan addresses the management of contamination present in various components of the quarry residuals operable unit (QROU) of the Weldon Spring site, which is located in St. Charles County, Missouri. The QROU consists of (1) residual waste at the quarry proper; (2) the Femme Osage Slough, Little Femme Osage Creek, and Femme Osage Creek; and (3) quarry groundwater located primarily north of the slough. Potential impacts to the St. Charles County well field downgradient of the quarry area are also being addressed as part of the evaluations for this operable unit. Remedial activities for the QROU will be conducted by the US Department of Energy (DOE) in accordance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended. As part of the remedial investigation/feasibility study (RI/FS) process required for the QROU under CERCLA, three major evaluation documents have been prepared to support cleanup decisions for this operable unit. decisions for this operable unit

Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

Directory of Open Access Journals (Sweden)

Cristina Marino Buslje

Full Text Available Identification of catalytic residues (CR is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI, and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls, combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

Science.gov (United States)

Marino Buslje, Cristina; Teppa, Elin; Di Doménico, Tomas; Delfino, José María; Nielsen, Morten

2010-11-04

Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

Strategy utilized for assessing baseline risks to human health from K-65 and metal oxide residues stored at the Fernald Site

Energy Technology Data Exchange (ETDEWEB)

Harmon, J.E. [FERMCO, Cincinnati, OH (United States). Fernald Environmental Management Project; Janke, R.C.

1995-04-01

The U.S. Department of Energy (DOE) is responsible for cleanup activities at the Fernald Environmental Management Project (FEMP) site in southwestern Ohio. The 425-hectare site consists of a former 55-hectare Production Area, an adjacent Waste Storage Area and various support facilities. From 1952 until 1989, the FEMP processed uranium into metallic {open_quotes}feed{close_quotes} materials for other DOE facilities in the nation`s defense program. In accordance with the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), the FEMP site is currently listed on the National Priorities List (NPL). To facilitate an expeditious cleanup effort, environmental issues associated with site cleanup are being managed under five operable units. This paper summarizes the risk assessment strategy employed to determine baseline human health risks associated with K-65 and metal oxide residues currently stored in Operable Unit 4. The K-65 and metal oxide residues were generated during the 1950s as a result of the extraction of uranium from uranium-bearing ores and concentrates. These residues are currently stored within Operable Unit 4 in concrete silos. Silos I and 2 contain approximately 6,120 cubic meters [m{sup 3}] (8,005 cubic yards [yd{sup 3}]) of K-65 residues, while silos 3 contains approximately 3890 m{sup 3} (5,080 yd{sup 3}) of cold metal oxides. These concrete silos are beyond their design life and require remedial action. The risk assessment conducted for Operable Unit 4 constitutes the first detailed human health risk assessment to be approved by the Environmental Protection Agency (EPA) for the CERCLA clean-up effort at the FEMP Site. This paper discusses the FEMP`s use of a Risk Information Quality Objective process in concert with the traditional risk assessment approach to determine baseline risk to human health and the environment posed by Operable Unit 4. A summary of the baseline risks to human health is also presented.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Science.gov (United States)

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Directory of Open Access Journals (Sweden)

Jung Hwa Lim

Full Text Available Here, we show that E2-EPF ubiquitin carrier protein (UCP elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196. A UCP mutant in which Cys118 was changed to alanine (UCPC118A did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Development of an on-site measurement method for residual stress in primary system piping of nuclear power plants

International Nuclear Information System (INIS)

Maekawa, Akira; Takahashi, Shigeru; Fujiwara, Masaharu

2014-01-01

In residual stress measurement for large-scale pipes and vessels in high radiation areas and highly contaminated areas of nuclear plants, it is difficult to bring the radioactivated pipes and vessels out of the areas as they are. If they can brought out, it is very burdensome to handle them for the measurement. Development of an on-site measurement method of residual stress which can be quickly applied and has sufficient measurement accuracy is desirable. In this study, a new method combining an electric discharge skim-cut method with a microscopic strain measurement method using markers was proposed to realize the on-site residual stress measurement on pipes in high radiation areas and highly contaminated areas. In the electric discharge skim-cut method, a boat-type sample is skimmed out of a pipe outer/inner surface using electric discharge machining and released residual stress is measured. The on-site measurement of residual stress by the method can be done using a small, portable electric discharge machine. In the microscopic strain measurement method using markers, the residual stress is estimated by microscopic measurement of the distance between markers after the stress release. The combination of both methods can evaluate the residual stress with the same accuracy as conventional methods offer and it can achieve reduction of radiation exposure in the measurement because the work is done simply and rapidly. In this study, the applicability of the electric discharge skim-cut method was investigated because the applicability of the microscopic strain measurement method using markers was confirmed previously. The experimental examination clarified the applicable conditions for the residual stress measurement with the same accuracy as the conventional methods. Furthermore, the electric discharge machining conditions using pure water as the machining liquid was found to eliminate the amount of liquid radioactive waste completely. (author)

Baseline risk assessment for the quarry residuals operable unit of the Weldon Spring Site, Weldon Spring, Missouri

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-02-01

The U.S. Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis. Cleanup of the site consists of several integrated components. The quarry residuals operable unit (QROU), consisting of the Weldon Spring quarry and its surrounding area, is one of four operable units being evaluated. In accordance with requirements of the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, DOE is conducting a remedial investigation/feasibility study (RI/FS) to determine the proper response to address various contaminated media that constitute the QROU. Specifically, the operable unit consists of the following areas and media: the residual material remaining at the Weldon Spring quarry after removal of the pond water and the bulk waste; groundwater underlying the quarry and surrounding area; and other media located in the surrounding vicinity of the quarry, including surface water and sediment at Femme Osage Slough, Little Femme Osage Creek, and Femme Osage Creek. An initial evaluation of conditions at the quarry area identified remaining data requirements needed to support the conceptual site exposure and hydrogeological models. These data requirements are discussed in the RI/FS work plan issued in January 1994. Soil contamination located at a property adjacent to the quarry, referred to as Vicinity Property 9 (VP9), was originally part of the scope of the QROU, as discussed in the work plan. However, a decision was subsequently made to remediate this vicinity property as part of cleanup activities for the chemical plant operable unit, as provided for in the Record of Decision (ROD). Remediation of VP9 was completed in early 1996. Hence, this baseline risk assessment (BRA) does not address VP9.

Baseline risk assessment for the quarry residuals operable unit of the Weldon Spring Site, Weldon Spring, Missouri

International Nuclear Information System (INIS)

1998-02-01

The U.S. Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis. Cleanup of the site consists of several integrated components. The quarry residuals operable unit (QROU), consisting of the Weldon Spring quarry and its surrounding area, is one of four operable units being evaluated. In accordance with requirements of the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, DOE is conducting a remedial investigation/feasibility study (RI/FS) to determine the proper response to address various contaminated media that constitute the QROU. Specifically, the operable unit consists of the following areas and media: the residual material remaining at the Weldon Spring quarry after removal of the pond water and the bulk waste; groundwater underlying the quarry and surrounding area; and other media located in the surrounding vicinity of the quarry, including surface water and sediment at Femme Osage Slough, Little Femme Osage Creek, and Femme Osage Creek. An initial evaluation of conditions at the quarry area identified remaining data requirements needed to support the conceptual site exposure and hydrogeological models. These data requirements are discussed in the RI/FS work plan issued in January 1994. Soil contamination located at a property adjacent to the quarry, referred to as Vicinity Property 9 (VP9), was originally part of the scope of the QROU, as discussed in the work plan. However, a decision was subsequently made to remediate this vicinity property as part of cleanup activities for the chemical plant operable unit, as provided for in the Record of Decision (ROD). Remediation of VP9 was completed in early 1996. Hence, this baseline risk assessment (BRA) does not address VP9

Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry.

Science.gov (United States)

Song, Hongjian; Olsen, Ole H; Persson, Egon; Rand, Kasper D

2014-12-19

Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Directory of Open Access Journals (Sweden)

Soares Alexei S

2007-11-01

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

Gamma exposure rate reduction and residual radium-226 concentrations resulting from decontamination activities conducted at the former uranium millsite in Shiprock, New Mexico

International Nuclear Information System (INIS)

Hans, J.M. Jr.; Hurst, T.L.

1981-01-01

Gamma radiation surveys and residual radium 226 soil samples were taken as part of the decontamination activities of the former Shiprock uranium mill site in New Mexico. In order to facilitate the decontamination activities, the mill site and its contaminated environs were divided into 6 major areas. Extensive data are presented in 2 appendices of the pre- and post-decontamination gamma ray exposure rates made on mill site, and of radium 226 concentrations in surface soil samples. A training program established on the mill site by the Navajo Engineering and Construction Authority is described

SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments

DEFF Research Database (Denmark)

Jessen, Leon Ivar; Hoof, Ilka; Lund, Ole

2013-01-01

Site does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set......) using a set of human immunodeficiency virus protease-inhibitor genotype–phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found...

Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

Directory of Open Access Journals (Sweden)

Ileana Corvo

Full Text Available A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2.Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS, we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL. Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3.These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage

Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447.

Science.gov (United States)

Yin, Ye; Liu, Pingsheng; Anderson, Richard G W; Sampson, Nicole S

2002-06-15

Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

International Nuclear Information System (INIS)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-01-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

SABER: a computational method for identifying active sites for new reactions.

Science.gov (United States)

Nosrati, Geoffrey R; Houk, K N

2012-05-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

Science.gov (United States)

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor

DEFF Research Database (Denmark)

Elling, C E; Thirstrup, K; Holst, Birgitte

1999-01-01

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor,...... as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.......Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor......, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated...

On using residual risk to assess the cost effectiveness and health protectiveness of remedy selection at superfund sites

International Nuclear Information System (INIS)

Katsumata, Peter T.; Kastenberg, William E.

1998-01-01

This article examines the importance of determining residual risk and its impact on remedy selection at Superfund Sites. Within this examination, risks are assessed using probabilistic models that incorporate the uncertainty and variability of the input parameters, and utilize parameter distributions based on current and applicable site-specific data. Monte Carlo methods are used to propagate these uncertainties and variabilities through the risk calculations resulting in a distribution for the estimate of both risk and residual risk. Such an approach permits an informed decision based on a broad information base which involves considering the entire uncertainty distribution of risk rather than a point estimate for each exposure scenario. Using the probabilistic risk estimates, with current and applicable site-specific data, alternative decisions regarding cleanup are obtained for two Superfund Sites

Three-dimensional modeling of the Nevada Test Site and vicinity from teleseismic p-wave residuals

International Nuclear Information System (INIS)

Monfort, M.E.; Evans, J.R.

1982-01-01

A teleseismic P-wave travel-time residual study is described which reveals the regional compressional-velocity structure of southern Nevada and neighboring parts of California to a depth of 280 km. During 1980, 98 teleseismic events were recorded at 53 sites. P-wave residuals were calculated relative to a network-wide average residual for each event and are displayed on maps of the stations for each of four event-azimuth quadrants. Fluctuations in these map-patterns of residuals with approach azimuth combined with results of linear, three-dimensional inversions of some 2887 residuals indicate the following characteristics of the velocity structure of the southern Nevada region: (1) a low-velocity body exists in the upper crust 50 km northeast of Beatty, Nevada, near the Miocene Timber Mountain-Silent Canyon caldera complex. Another highly localized low-velocity anomaly occurs near the southwest corner of the Nevada Test Site (NTS). These two anomalies seem to be part of a low-velocity trough extending from Death Valley, California, to about 50 km north of NTS; (2) there is a high-velocity body in the mantle between 81 and 131 km deep centered about 10 km north of the edge of the Timber Mountain caldera; (3) a broad low-velocity body is delineated between 81 and 131 km deep centered about 30 km north of Las Vegas; (4) there is a monotonic increase in travel-time delays from west to east across the region, probably indicating an eastward decrease in velocity, and lower than average velocities in southeastern Nevada below 31 km; and (5) considerable complexity in three-dimensional velocity structure exists in this part of the southern Great Basin. Inversions of teleseismic P-wave travel-time residuals were also performed on data from 12 seismometers in the immediate vicinity of the NTS to make good use of the closer station spacing in that area

Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

Science.gov (United States)

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

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase

International Nuclear Information System (INIS)

Nishino, T.; Nishino, T.

1987-01-01

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O 2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [ 14 C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14 C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine

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

NARCIS (Netherlands)

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

2008-01-01

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

Sustainable System for Residual Hazards Management

International Nuclear Information System (INIS)

Kevin M. Kostelnik; James H. Clarke; Jerry L. Harbour

2004-01-01

Hazardous, radioactive and other toxic substances have routinely been generated and subsequently disposed of in the shallow subsurface throughout the world. Many of today's waste management techniques do not eliminate the problem, but rather only concentrate or contain the hazardous contaminants. Residual hazards result from the presence of hazardous and/or contaminated material that remains on-site following active operations or the completion of remedial actions. Residual hazards pose continued risk to humans and the environment and represent a significant and chronic problem that require continuous long-term management (i.e. >1000 years). To protect human health and safeguard the natural environment, a sustainable system is required for the proper management of residual hazards. A sustainable system for the management of residual hazards will require the integration of engineered, institutional and land-use controls to isolate residual contaminants and thus minimize the associated hazards. Engineered controls are physical modifications to the natural setting and ecosystem, including the site, facility, and/or the residual materials themselves, in order to reduce or eliminate the potential for exposure to contaminants of concern (COCs). Institutional controls are processes, instruments, and mechanisms designed to influence human behavior and activity. System failure can involve hazardous material escaping from the confinement because of system degradation (i.e., chronic or acute degradation) or by external intrusion of the biosphere into the contaminated material because of the loss of institutional control. An ongoing analysis of contemporary and historic sites suggests that the significance of the loss of institutional controls is a critical pathway because decisions made during the operations/remedial action phase, as well as decisions made throughout the residual hazards management period, are key to the long-term success of the prescribed system. In fact

Criteria for the restoration of mining residues in Germany

International Nuclear Information System (INIS)

Kraus, W.; Ettenhuber, E.; Gehrcke, K.; Przyborowski, S.

2000-01-01

Residues from uranium mines and mills and from the mining of silver, tin, cobalt, nickel and other ores, as well as of coal mineralized with uranium, are situated in densely populated regions of Germany. Social and political pressure required an urgent investigation and evaluation of these residues in order to identify relevant residues which could not be disregarded from the radiation protection point of view. There were two categories of residues. First, for huge former uranium mining and milling sites, the original owner could be made liable for restoration. A large Federal rehabilitation programme for the Wismut sites was started immediately after the political change in the former East Germany in 1990 and was based on radiological as well as on social and economic concerns. Secondly, for a large number of smaller residues, sometimes dating back to the middle ages, an evaluation of their radiological relevance was necessary before decisions could be taken on the justification of a restoration. This was the objective of a Federal programme on registration, investigation and evaluation of mining residues. Up to now only minor remedial activities have been carried out in cases where an urgent need had been detected. Criteria developed by the German Commission on radiological protection (SSK) were applied for the evaluation of the residues. The primary criterion for the justification of a restoration was an annual individual effective dose of 1 mSv for all exposure pathways except for the inhalation of radon. For inhalation of radon, the primary criterion for justification was a long term average outdoor radon concentration of 50 Bq/m 3 caused by the residues. Both levels were taken in addition to the natural background radiation level at a given site. These criteria were based on the upper end of the 'normal' range of naturally occurring exposure or concentration levels. SSK established reference levels in measurable quantities (activity concentration in soil

Transport and Application of Heat-Activated Persulfate for In-situ Chemical Oxidation of Residual Trichloroethylene

Science.gov (United States)

Quig, L.; Johnson, G. R.

2015-12-01

Persulfate ISCO has been shown to treat a wide range of contaminants. While persulfate ISCO can be tailored to site and pollutant specific characteristics (e.g., activation via energy or catalysis), thermal activation of persulfate is particularly promising as it can be easily controlled and requires no additional reagents. A mechanistic study of the physical and chemical processes controlling the effectiveness of this remedial approach is not well documented in the literature with much therein focused on reactions in batch systems. The purpose of this research was twofold. Initial studies characterized the overall transport behavior of unactivated and thermally-activated persulfate (20, 60, and 90°C) in one-dimensional soil column systems. Finally, experiments were conducted to investigate persulfate ISCO as a remedial approach for residual-phase trichloroethylene (TCE). At all activation temperatures investigated, persulfate exhibited ideal transport behavior in miscible displacement experiments. Moment analysis of persulfate ion breakthrough curves indicated negligible interaction of persulfate with the natural sandy material. Persulfate ISCO for residual-phase TCE was characterized at two flow rates, 0.2 mL/min and 0.5 mL/min, resulting in two degrees of persulfate activation, 39.5% and 24.6%, respectively. Both ISCO soil column systems showed an initial, long-term plateau in effluent TCE concentrations indicating steady-state dissolution of pure phase TCE. Observed effluent concentrations decreased after 75 and 100 pore volumes (normalized for the measured residual NAPL fraction) compared to 110 pore volumes in the control study. Pseudo first-order reaction rate constants for the decreasing TCE concentrations equaled 0.063/hr and 0.083/hr, respectively, compared to 0.041/hr for the control. Moment analysis of the complete dissolution of TCE in the persulfate/activated persulfate remediation systems indicated approximately 33% oxidation of TCE mass present. By

Derivation of cesium-137 residual radioactive material guidelines for the Peek Street site, Schenectady, New York

International Nuclear Information System (INIS)

Jones, L.; Nimmagadda, M.; Yu, C.

1992-01-01

Residual radioactive material guidelines for cesium-137 were derived for the Peek rk. The derivation was based on the requirement that the Street site in Schenectady, New York. The derivation was based on the requirement that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works in the immediate vicinity of the Peek Street site should not exceed a dose of 100 mrem/yr following remedial action. The US Department of Energy (DOE) residual radioactive material guideline computer code, RESRAD was used in this evaluation. Three potential scenarios were considered for the site on the assumption that for a period of 1,000 years following remedial action, the site wig be utilized without radiological restrictions. The scenarios vary with regard to use of the site, time spent at the site, and sources of food consumed. Results indicate that the basic dose limit of 100 mrem/yr will not be exceeded for cesium-137 within 1,000 years, provided that the soil concentration of cesium-137 at the Peek Street site does not exceed the following levels: 98 pCi/g for Scenario A (industrial worker: the expected scenario), 240 pCi/g for Scenario B (recreationist: a plausible scenario), and 34 pCi/g for Scenario C (resident farmer ingesting food produced in the decontaminated area: a plausible scenario)

Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

Directory of Open Access Journals (Sweden)

Tshidi Tsibane

Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

Characterization Report on Sand, Slag, and Crucible Residues and on Fluoride Residues

International Nuclear Information System (INIS)

Murray, A.M.

1999-01-01

This paper reports on the chemical characterization of the sand, slag, and crucible (SS and C) residues and the fluoride residues that may be shipped from the Rocky Flats Environmental Technology Site (RFETS) to Savannah River Site (SRS)

Identification of Key Residues for Enzymatic Carboxylate Reduction

Directory of Open Access Journals (Sweden)

Holly Stolterfoht

2018-02-01

Full Text Available Carboxylate reductases (CARs, E.C. 1.2.1.30 generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

A Simple Surface-Enhanced Raman Spectroscopic Method for on-Site Screening of Tetracycline Residue in Whole Milk

Directory of Open Access Journals (Sweden)

Sagar Dhakal

2018-02-01

Full Text Available Therapeutic and subtherapeutic use of veterinary drugs has increased the risk of residue contamination in animal food products. Antibiotics such as tetracycline are used for mastitis treatment of lactating cows. Milk expressed from treated cows before the withdrawal period has elapsed may contain tetracycline residue. This study developed a simple surface-enhanced Raman spectroscopic (SERS method for on-site screening of tetracycline residue in milk and water. Six batches of silver colloid nanoparticles were prepared for surface enhancement measurement. Milk-tetracycline and water-tetracycline solutions were prepared at seven concentration levels (1000, 500, 100, 10, 1, 0.1, and 0.01 ppm and spiked with silver colloid nanoparticles. A 785 nm Raman spectroscopic system was used for spectral measurement. Tetracycline vibrational modes were observed at 1285, 1317 and 1632 cm−1 in water-tetracycline solutions and 1322 and 1621 cm−1 (shifted from 1317 and 1632 cm−1, respectively in milk-tetracycline solutions. Tetracycline residue concentration as low as 0.01 ppm was detected in both the solutions. The peak intensities at 1285 and 1322 cm−1 were used to estimate the tetracycline concentrations in water and milk with correlation coefficients of 0.92 for water and 0.88 for milk. Results indicate that this SERS method is a potential tool that can be used on-site at field production for qualitative and quantitative detection of tetracycline residues.

A Simple Surface-Enhanced Raman Spectroscopic Method for on-Site Screening of Tetracycline Residue in Whole Milk.

Science.gov (United States)

Dhakal, Sagar; Chao, Kuanglin; Huang, Qing; Kim, Moon; Schmidt, Walter; Qin, Jianwei; Broadhurst, C Leigh

2018-02-01

Therapeutic and subtherapeutic use of veterinary drugs has increased the risk of residue contamination in animal food products. Antibiotics such as tetracycline are used for mastitis treatment of lactating cows. Milk expressed from treated cows before the withdrawal period has elapsed may contain tetracycline residue. This study developed a simple surface-enhanced Raman spectroscopic (SERS) method for on-site screening of tetracycline residue in milk and water. Six batches of silver colloid nanoparticles were prepared for surface enhancement measurement. Milk-tetracycline and water-tetracycline solutions were prepared at seven concentration levels (1000, 500, 100, 10, 1, 0.1, and 0.01 ppm) and spiked with silver colloid nanoparticles. A 785 nm Raman spectroscopic system was used for spectral measurement. Tetracycline vibrational modes were observed at 1285, 1317 and 1632 cm -1 in water-tetracycline solutions and 1322 and 1621 cm -1 (shifted from 1317 and 1632 cm -1 , respectively) in milk-tetracycline solutions. Tetracycline residue concentration as low as 0.01 ppm was detected in both the solutions. The peak intensities at 1285 and 1322 cm -1 were used to estimate the tetracycline concentrations in water and milk with correlation coefficients of 0.92 for water and 0.88 for milk. Results indicate that this SERS method is a potential tool that can be used on-site at field production for qualitative and quantitative detection of tetracycline residues.

Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes.

Directory of Open Access Journals (Sweden)

Benjamin R Jack

2016-05-01

Full Text Available 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.

The behaviour of residual contaminants at a former station site, Antarctica

International Nuclear Information System (INIS)

Webster, Jenny; Webster, Kerry; Nelson, Peter; Waterhouse, Emma

2003-01-01

Minor contamination by metals, phosphorus, and fuel products were found at a former research station site in Antarctica. - In 1994, New Zealand's only mainland Antarctic base, Vanda Station, was removed from the shores of Lake Vanda, in the McMurdo Dry Valleys region of southern Victoria Land, Antarctica. Residual chemical contamination of the station site has been identified, in the form of discrete fuel spills, locally elevated Pb, Zn, Ag and Cd concentrations in soil and elevated Cu, Ni, Co and phosphate concentrations in suprapermafrost fluids in a gully formerly used for domestic washing water disposal. Pathways for contaminant transfer to Lake Vanda, potential environmental impacts and specific remediation/monitoring options are considered. While some contaminants (particularly Zn) could be selectively leached from flooded soil, during a period of rising lake level, the small area of contaminated soils exposed and low level of contamination suggests that this would not adversely affect either shallow lake water quality or the growth of cyanobacteria. Phosphate-enhanced growth of the latter may, however, be a visible consequence of the minor contamination occurring at this site

Calcination/dissolution residue treatment

International Nuclear Information System (INIS)

Knight, R.C.; Creed, R.F.; Patello, G.K.; Hollenberg, G.W.; Buehler, M.F.; O'Rourke, S.M.; Visnapuu, A.; McLaughlin, D.F.

1994-09-01

Currently, high-level wastes are stored underground in steel-lined tanks at the Hanford site. Current plans call for the chemical pretreatment of these wastes before their immobilization in stable glass waste forms. One candidate pretreatment approach, calcination/dissolution, performs an alkaline fusion of the waste and creates a high-level/low-level partition based on the aqueous solubilities of the components of the product calcine. Literature and laboratory studies were conducted with the goal of finding a residue treatment technology that would decrease the quantity of high-level waste glass required following calcination/dissolution waste processing. Four elements, Fe, Ni, Bi, and U, postulated to be present in the high-level residue fraction were identified as being key to the quantity of high-level glass formed. Laboratory tests of the candidate technologies with simulant high-level residues showed reductive roasting followed by carbonyl volatilization to be successful in removing Fe, Ni, and Bi. Subsequent bench-scale tests on residues from calcination/dissolution processing of genuine Hanford Site tank waste showed Fe was separated with radioelement decontamination factors of 70 to 1,000 times with respect to total alpha activity. Thermodynamic analyses of the calcination of five typical Hanford Site tank waste compositions also were performed. The analyses showed sodium hydroxide to be the sole molten component in the waste calcine and emphasized the requirement for waste blending if fluid calcines are to be achieved. Other calcine phases identified in the thermodynamic analysis indicate the significant thermal reconstitution accomplished in calcination

Triazacyclophane (TAC)-scaffolded histidine and aspartic acid residues as mimics of non-heme metalloenzyme active sites

NARCIS (Netherlands)

Albada, H.B.; Soulimani, F.; Jacobs, H.J.F.; Versluis, C.; Weckhuysen, B.M.; Liskamp, R.M.J.

2012-01-01

We describe the synthesis and coordination behaviour to copper(II) of two close structural triazacyclophane-based mimics of two often encountered aspartic acid and histidine containing metalloenzyme active sites. Coordination of these mimics to copper(I) and their reaction with molecular oxygen

Derivation of residual radioactive material guidelines for uranium in soil at the Former Associate Aircraft Tool and Manufacturing Company Site, Fairfield, Ohio

International Nuclear Information System (INIS)

Faillace, E.R.; Nimmagadda, M.; Yu, C.

1995-01-01

Residual radioactive material guidelines for uranium in soil were derived for the former Associate Aircraft Tool and Manufacturing Company site in Fairfield, Ohio. This site has been identified for remedial action under the U.S. Department of Energy's (DOE's) Formerly Utilized Sites Remedial Action Program (FUSRAP). Single-nuclide and total-uranium guidelines were derived on the basis of the requirement that, after remedial action, the 50-year committed effective dose equivalent to a hypothetical individual living or working in the immediate vicinity of the site should not exceed (1) 30 mrem/yr for the current-use and likely future-use scenarios or (2) 100 mrem/yr for less likely future-use scenarios. The DOE residual radioactive material (RESRAD) computer code, which implements the methodology described in the DOE manual for establishing residual radioactive material guidelines, was used in this evaluation

Networks of High Mutual Information Define the Structural Proximity of Catalytic Sites: Implications for Catalytic Residue Identification

DEFF Research Database (Denmark)

Buslje, Cristina Marino; Teppa, Elin; Di Doménico, Tomas

2010-01-01

other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown.......90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function...

Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

DEFF Research Database (Denmark)

Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian

2014-01-01

, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β-casein on HRP disappears when pH is below the isoelectric point of β-casein. It was also proven by light scattering studies that β...... proteins. Incubating HRP (0.1 mg mL-1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL-1 β-casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration......-casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries...

Feasibility study for remedial action for the Quarry Residuals Operable Unit at the Weldon Spring Site, Weldon Spring, Missouri

International Nuclear Information System (INIS)

1998-03-01

The U.S. Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, which is located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis (Figure 1.1). Cleanup of the Weldon Spring site consists of several integrated components. The quarry residuals operable unit (QROU) is one of four operable units being evaluated. In accordance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, a remedial investigation/feasibility study (RI/FS) is being conducted to evaluate conditions and potential responses for the following areas and/or media that constitute the QROU: (1) the residual material (soil and sediment) remaining at the Weldon Spring quarry after removal of the bulk waste (about 11 million L [3 million gal] of uranium-contaminated ponded water was also addressed previous to bulk waste removal); (2) other media located in the surrounding vicinity of the quarry, including adjacent soil, surface water, and sediment in Femme Osage Slough and several creeks; and (3) quarry groundwater located primarily north of Femme Osage Slough. Potential impacts to the St. Charles County well field downgradient of the quarry area are also being addressed as part of QROU RI/FS evaluations. For remedial action sites, it is DOE policy to integrate values associated with the National Environmental Policy Act (NEPA) into the CERCLA decision-making process. The analyses contained herein address NEPA values as appropriate to the actions being considered for the QROU. A work plan summarizing initial site conditions and providing conceptual site hydrogeological and exposure models was published in January 1994. The RI and baseline risk assessment (BRA) reports have been completed. The RI discusses in detail the nature and extent and the fate and transport of contamination at the quarry area

Feasibility study for remedial action for the Quarry Residuals Operable Unit at the Weldon Spring Site, Weldon Spring, Missouri

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-03-01

The U.S. Department of Energy (DOE) is conducting cleanup activities at the Weldon Spring site, which is located in St. Charles County, Missouri, about 48 km (30 mi) west of St. Louis (Figure 1.1). Cleanup of the Weldon Spring site consists of several integrated components. The quarry residuals operable unit (QROU) is one of four operable units being evaluated. In accordance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), as amended, a remedial investigation/feasibility study (RI/FS) is being conducted to evaluate conditions and potential responses for the following areas and/or media that constitute the QROU: (1) the residual material (soil and sediment) remaining at the Weldon Spring quarry after removal of the bulk waste (about 11 million L [3 million gal] of uranium-contaminated ponded water was also addressed previous to bulk waste removal); (2) other media located in the surrounding vicinity of the quarry, including adjacent soil, surface water, and sediment in Femme Osage Slough and several creeks; and (3) quarry groundwater located primarily north of Femme Osage Slough. Potential impacts to the St. Charles County well field downgradient of the quarry area are also being addressed as part of QROU RI/FS evaluations. For remedial action sites, it is DOE policy to integrate values associated with the National Environmental Policy Act (NEPA) into the CERCLA decision-making process. The analyses contained herein address NEPA values as appropriate to the actions being considered for the QROU. A work plan summarizing initial site conditions and providing conceptual site hydrogeological and exposure models was published in January 1994. The RI and baseline risk assessment (BRA) reports have been completed. The RI discusses in detail the nature and extent and the fate and transport of contamination at the quarry area.

On-site and off-site activities

International Nuclear Information System (INIS)

Martin, H.D.

1986-01-01

Design principles for NPP training programs. Effects of NPP contracts. Effects of domestic industrial activities. The role of international bodies. Requirements for on-site training. Training abroad, technical, financial and social aspects. Training center on-site, an evaluation. (orig.)

Assessment of alternatives for long-term management of uranium ore residues and contaminated soils located at DOE's Niagara Falls Storage Site

International Nuclear Information System (INIS)

Merry-Libby, P.

1985-01-01

About 11,000 m 3 of uranium ore residues and 180,000 m 3 of slightly contaminated soils (wastes) are consolidated within a diked containment area at the Niagara Falls Storage Site located about 30 km north of Buffalo, New York. The residues account for less than 6% of the total volume of contaminated materials but almost 99% of the radioactivity. The average radium-226 concentration in the residues is 67,000 pCi/g. The US Department of Energy is considering several alternatives for long-term management of the wastes and residues, including: improvement of the containment at NFSS, modification of the form of the residues, management of the residues separately from the wastes, management of the wastes and residues at another humid site (Oak Ridge, Tennessee) or an arid site (Hanford, Washington), and dispersal of the wastes in the ocean. Potential radiological risks associated with implementation of any of the alternatives are expected to be smaller than the nonradiological risks of occupational and transportation-related injuries and deaths. Dispersal of the slightly contaminated wastes in the ocean is not expected to result in any significant radiological risk to humans. The residues and wastes will remain hazardous for thousands of years. After controls cease, the radioactive materials will eventually be dispersed in the environment. Loss of the earthen covers over the buried materials is predicted to occur from several hundred to more than two million years, depending primarily on the use of the land surface. Groundwater will eventually be contaminated in all alternatives; however, the groundwater pathway is relatively insignificant with respect to radiological risks to the general population. 2 references, 2 figures, 6 tables

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

Science.gov (United States)

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

Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Su, Hua-Poo; Yan, Youwei; Prasad, G. Sridhar; Smith, Robert F.; Daniels, Christopher L.; Abeywickrema, Pravien D.; Reid, John C.; Loughran, H. Marie; Kornienko, Maria; Sharma, Sujata; Grobler, Jay A.; Xu, Bei; Sardana, Vinod; Allison, Timothy J.; Williams, Peter D.; Darke, Paul L.; Hazuda, Daria J.; Munshi, Sanjeev (Merck)

2010-09-02

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

Science.gov (United States)

Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João M; Belizário, José E

2017-12-01

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD 198 ↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (k cat /K M ) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

Directory of Open Access Journals (Sweden)

Jiao Chen

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

The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

DEFF Research Database (Denmark)

Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

2016-01-01

Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN...

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

Directory of Open Access Journals (Sweden)

Ueno Keisuke

2012-04-01

Full Text Available Abstract 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.

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

Science.gov (United States)

Basu, S; Mandal, C; Allen, A K

1988-01-01

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin. Images Fig. 3. PMID:3140796

Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM.

Science.gov (United States)

Khosravi, Mojtaba; Bringolf, Fanny; Röthlisberger, Silvan; Bieringer, Maria; Schneider-Schaulies, Jürgen; Zurbriggen, Andreas; Origgi, Francesco; Plattet, Philippe

2016-02-01

Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces ("front" and "back"). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. A complete understanding of the measles virus and canine distemper virus

Role of cysteine residues in the structure, stability, and alkane producing activity of cyanobacterial aldehyde deformylating oxygenase.

Directory of Open Access Journals (Sweden)

Yuuki Hayashi

Full Text Available Aldehyde deformylating oxygenase (AD is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

Endosulfan residues in fish from the MUDA rice agroecosystem

International Nuclear Information System (INIS)

Nashriyah Mat; Sabri Junoh; Nuriati Nurdin

2002-01-01

A study was carried out in the recycled and the non-recycled areas of the Muda rice agroecosystem to determine the residue level of endosulfan in rice field fish. Snakehead or haruan (Channa striata), climbing perch or puyu (Anabas testudineus), walking catfish or keli (Clariasspp.) and snakeskin gouramy or sepat siam (Trichogasterpectoralis) were sampled and the tissues analysed using Gas Liquid Chromatography. Endosulfan sulphate metabolite was a ubiquitous residue in all fish at levels ranging from 0.0065 μg/g to 0.9837 μg/g of tissue wet weight whilst α-isomer was detected at a much lower concentration followed by β-isomer. In Channa striata, endosulfan total residue (α+β+sulphate) was accumulated in fish tissues sampled at ACRBD4 (non-recycled) site followed by Blok 14 (recycled) site. Endosulfan residue concentration did not coincide with water recycling practice nor changes in EROD enzyme activity. (Author)

Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

Directory of Open Access Journals (Sweden)

Kevin A James

Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

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

Science.gov (United States)

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

2015-03-01

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

A study on the neoasozine residues in rice grain by neutron activation method

International Nuclear Information System (INIS)

Kim, Y.H.; Lee, K.J.; Lee, S.R.

1981-01-01

Residues of neoasozine in rice grain were determined by neutron activation and colorimetric techniques. Twice application of the chemical before flowering did not lead to any increased residue level while 4-times application resulted in significant increase in the residue level up to 0.54 - 0.75 mg As 2 O 3 /kg. The partition ratio of arsenic residues into polished rice grain and bran was 73 : 27 in 100 % polishing while most of the residues in the bran was transferred to oil cake fraction during solvent extraction, reaching up to 2.9 mg As 2 O 3 /kg. The neutron activation technique was advantageous because of its high sensitivity and the smaller sample amounts required for analysis. (author)

Testing in support of on-site storage of residues in the Pipe Overpack Container

International Nuclear Information System (INIS)

Ammerman, D.J.; Bobbe, J.G.; Arviso, M.

1997-02-01

The disposition of the large back-log of plutonium residues at the Rocky Flats Environmental Technology Site (Rocky Flats) will require interim storage and subsequent shipment to a waste repository. Current plans call for disposal at the Waste Isolation Pilot Plant (WIPP) and the transportation to WIPP in the TRUPACT-II. The transportation phase will require the residues to be packaged in a container that is more robust than a standard 55-gallon waste drum. Rocky Flats has designed the Pipe Overpack Container to meet this need. It is desirable to use this same waste packaging for interim on-site storage in non-hardened buildings. To meet the safety concerns for this storage the Pipe Overpack Container has been subjected to a series of tests at Sandia National Laboratories in Albuquerque, New Mexico. In addition to the tests required to qualify the Pipe Overpack Container as a waste container for shipment in the TRUPACT-II several tests were performed solely for the purpose of qualifying the container for interim storage. This report will describe these tests and the packages response to the tests. 12 figs., 3 tabs

Comparison of the level of residual coagulant activity in different cheese varieties.

Science.gov (United States)

Bansal, Nidhi; Fox, Patrick F; McSweeney, Paul L H

2009-08-01

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

Science.gov (United States)

Flückiger, Rudolf; Cocuzzi, Enzo; Nagaraj, Ram H; Shoham, Menachem; Kern, Timothy S; Medof, M Edward

2018-01-01

Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K 125 adjacent to K 126 , K 127 at the junction of CCPs2-3 and spatially near R 96 , and R 100 , all identified as being critical for DAF's function. Functional analyses of glucose or ribose treated DAF protein showed profound loss of its regulatory activity. The data argue that de-regulated activation of systemic complement and de-regulated activation of T cells and leukocytes could result from non-enzymatic glycation of DAF. Copyright © 2017. Published by Elsevier Ltd.

DOE site performance assessment activities

International Nuclear Information System (INIS)

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

Determination of Activated Carbon Residual Life using a Microwave Cavity Resonator

International Nuclear Information System (INIS)

Mason, A; Wylie, S; Shaw, A; Al-Shamma'a, A I; Thomas, A; Keele, H

2011-01-01

This paper presents the continuation of work conducted jointly between Dstl and LJMU. This unique body of work has been, largely, concerned with detecting the residual life of high performance filter materials using electromagnetic (EM) waves within a resonant cavity. Past work has considered both HEPA [1] and ASZM-TEDA[2] activated carbon filter materials. This paper continues the later work, considering the response of ASZM-TEDA activated carbon through the co-ageing of two distinct batches of the material. The paper briefly introduces activated carbon, discusses theory relevant to the work and the methodology used for investigation. A comprehensive set of results is included which seek to validate this technique for determining the residual lifespan of activated carbon.

Derivation of guidelines for uranium residual radioactive material in soil at the B ampersand T Metals Company site, Columbus, Ohio

International Nuclear Information System (INIS)

Kamboj, S.; Nimmagadda, Mm.; Yu, C.

1996-01-01

Guidelines for uranium residual radioactive material in soil were derived for the B ampersand T Metals Company site in Columbus, Ohio. This site has been identified for remedial action under the US Department of Energy's (DOE's) Formerly Utilized Sites Remedial Action Program (FUSRAP). Single-nuclide and total-uranium guidelines were derived on the basis of the requirement that following remedial action, the 50-year committed effective dose equivalent to a hypothetical individual living or working in the immediate vicinity of the site should not exceed a dose constraint of 30 mrem/yr for the current use and likely future use scenarios or a dose limit of 100 n-mrem/yr for less likely future use scenarios. The DOE residual radioactive material guideline computer code, RESRAD, was used in this evaluation. RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines. Three scenarios were considered; each assumed that for a period of 1,000 years following remedial action, the site would be used without radiological restrictions. The three scenarios varied with regard to the type of site use, time spent at the site by the exposed individual, and sources of food and water consumed. The evaluations indicate that the dose constraint of 30 mrem/yr would not be exceeded for uranium (including uranium-234, uranium-235, and uranium-238) within 1,000 years, provided that the soil concentration of total uranium (uranium-234, uranium-235, and uranium-238) at the B ampersand T Metals site did not exceed 1, I 00 pCi/g for Scenario A (industrial worker, current use) or 300 pCi/g for Scenario B (resident with municipal water supply, a likely future use). The dose limit of 100 mrem/yr would not be exceeded at the site if the total uranium concentration of the soil did not exceed 880 pCi/g for Scenario C (resident with an on-site water well, a plausible but unlikely future use)

Identification of residues important for the activity of Haloferax volcanii AglD, a component of the archaeal N-glycosylation pathway.

Science.gov (United States)

Kaminski, Lina; Eichler, Jerry

2010-05-06

In Haloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in an Hfx. volcanii aglD deletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

Rebaselining of the plutonium residue elimination project at Rocky Flats Environmental Technology Site

International Nuclear Information System (INIS)

Sailor, W.C.; Catlett, D.S.; Burns, T.P.

1997-01-01

Systems Engineering and Value Engineering principles were put into practice in rebaselining the Pu Residue Stabilization and Elimination Project at the Rocky Flats Environmental Technology Site. Tradeoff studies were conducted as to how to best rebaseline the system under the new Safeguards Termination Limits (STSs) issued by the Department of Energy. Through the use of a computerized database, the means by which Stakeholder values and other high-level requirements have been included in the tradeoff studies were documented. 13 refs., 2 figs., 1 tab

Rebaselining of the plutonium residue elimination project at Rocky Flats Environmental Technology Site

Energy Technology Data Exchange (ETDEWEB)

Sailor, W.C.; Catlett, D.S.; Burns, T.P. [and others

1997-03-01

Systems Engineering and Value Engineering principles were put into practice in rebaselining the Pu Residue Stabilization and Elimination Project at the Rocky Flats Environmental Technology Site. Tradeoff studies were conducted as to how to best rebaseline the system under the new Safeguards Termination Limits (STSs) issued by the Department of Energy. Through the use of a computerized database, the means by which Stakeholder values and other high-level requirements have been included in the tradeoff studies were documented. 13 refs., 2 figs., 1 tab.

Physical stability comparisons of IgG1-Fc variants: effects of N-glycosylation site occupancy and Asp/Gln residues at site Asn 297.

Science.gov (United States)

Alsenaidy, Mohammad A; Okbazghi, Solomon Z; Kim, Jae Hyun; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

2014-06-01

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

The Effect of Kaffir Lime Leaves Distillation Residue Oleoresin Concentration on Active Paper Packaging Characteristics

Science.gov (United States)

Kawiji; Utami, R.; Ulum, S.; Khasanah, L. U.; Manuhara, G. J.; Atmaka, W.

2018-03-01

Oleoresin of kaffir lime leaves distillation residue still contains some active compounds such as Citronellal, β-Citronellol, and Linalool which potential to incorporated on the active paper packaging. The purposes of this study were to determine the effect of kaffir lime leaves distillation residue oleoresin concentration on the physical characteristics, sensory characteristics, and antimicrobial activity of the active paper packaging incorporated with kaffir lime leaves distillation residue oleoresin and to determine the functional groups of active paper packaging. The concentration of kaffir lime leaves distillation residue oleoresin were varied at 0%, 2%, 4% and 6%. The result showed that the addition of kaffir lime leaves distillation residue oleoresin increased the thickness and moisture content of the paper and decreased the tensile strengths and folding endurances of active paper packaging. The microbial inhibition tends to increase along with the higher oleoresin concentration addition. Aromatic CH group were found at a wavelength of 897.90 cm-1 of on paper packaging with 2% oleoresin indicated as functional aromatic functional group allegedly obtained from the kaffir lime leaves oleoresin.

Studies on recycling and utilization of spent catalysts. Preparation of active hydrodemetallization catalyst compositions from spent residue hydroprocessing catalysts

Energy Technology Data Exchange (ETDEWEB)

Marafi, Meena; Stanislaus, Antony [Petroleum Refining Department, Petroleum Research and Studies Center, Kuwait Institute for Scientific Research, P.O. Box 24885, Safat (Kuwait)

2007-02-15

Spent catalysts form a major source of solid wastes in the petroleum refining industries. Due to environmental concerns, increasing emphasis has been placed on the development of recycling processes for the waste catalyst materials as much as possible. In the present study the potential reuse of spent catalysts in the preparation of active new catalysts for residual oil hydrotreating was examined. A series of catalysts were prepared by mixing and extruding spent residue hydroprocessing catalysts that contained C, V, Mo, Ni and Al{sub 2}O{sub 3} with boehmite in different proportions. All prepared catalysts were characterized by chemical analysis and by surface area, pore volume, pore size and crushing strength measurements. The hydrodesulfurization (HDS) and hydrodemetallization (HDM) activities of the catalysts were evaluated by testing in a high pressure fixed-bed microreactor unit using Kuwait atmospheric residue as feed. A commercial HDM catalyst was also tested under similar operating conditions and their HDS and HDM activities were compared with that of the prepared catalysts. The results revealed that catalyst prepared with addition of up to 40 wt% spent catalyst to boehmite had fairly high surface area and pore volume together with large pores. The catalyst prepared by mixing and extruding about 40 wt% spent catalyst with boehmite was relatively more active for promoting HDM and HDS reactions than a reference commercial HDM catalyst. The formation of some kind of new active sites from the metals (V, Mo and Ni) present in the spent catalyst is suggested to be responsible for the high HDM activity of the prepared catalyst. (author)

Explaining an Unusually Fast Parasitic Enzyme: Folate Tail-Binding Residues Dictate Substrate Positioning and Catalysis in Cryptosporidium hominis Thymidylate Synthase

Energy Technology Data Exchange (ETDEWEB)

Martucci,W.; Vargo, M.; Anderson, K.

2008-01-01

The essential enzyme TS-DHFR from Cryptosporidium hominis undergoes an unusually rapid rate of catalysis at the conserved TS domain, facilitated by two nonconserved residues, Ala287 and Ser290, in the folate tail-binding region. Mutation of these two residues to their conserved counterparts drastically affects multiple steps of the TS catalytic cycle. We have determined the crystal structures of all three mutants (A287F, S290G, and A287F/S290G) in complex with active site ligands dUMP and CB3717. The structural data show two effects of the mutations: an increased distance between the ligands in the active site and increased flexibility of the folate ligand in the partially open enzyme state that precedes conformational change to the active catalytic state. The latter effect is able to be rescued by the mutants containing the A287F mutation. In addition, the conserved water network of TS is altered in each of the mutants. The structural results point to a role of the folate tail-binding residues in closely positioning ChTS ligands and restricting ligand flexibility in the partially open state to allow for a rapid transition to the active closed state and enhanced rate of catalysis. These results provide an explanation on how folate tail-binding residues at one end of the active site affect long-range interactions throughout the TS active site and validate these residues as targets for species-specific drug design.

Build Rocky Flats Environmental Technology site production prototype modular treatment system for stand alone core capability for residue unpack, sort, assay, repack

International Nuclear Information System (INIS)

Hildner, R.A.; Zygmunt, S.J.

1997-01-01

This document describes a portable and modular suit of equipment that upfront and near-term accomplishes a sorting process that documents and removes Rocky Flats Environmental Technology Site (RFETS) residue and waste from site inventory

Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A

DEFF Research Database (Denmark)

Kont, Riin; Kari, Jeppe; Borch, Kim

2016-01-01

systems. TrCel7A consists of catalytic domain (CD) and a smaller carbohydrate binding module (CBM) connected through the glycosylated linker peptide. A tunnel shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two...... to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient......Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme...

Contaminant Release from Residual Waste in Single Shell Tanks at the Hanford Site, Washington, USA - 9276

International Nuclear Information System (INIS)

Cantrell, Kirk J.; Krupka, Kenneth M.; Deutsch, William J.; Lindberg, Michael J.

2009-01-01

Determinations of elemental and solid-phase compositions, and contaminant release studies have been applied in an ongoing study of residual tank wastes (i.e., waste remaining after final retrieval operations) from five of 149 underground single-shell storage tanks (241-C-103, 241-C-106, 241-C-202, 241-C-203, and 241-S-112) at the U.S. Department of Energy's Hanford Site in Washington State. This work is being conducted to support performance assessments that will be required to evaluate long-term health and safety risks associated with tank site closure. The results of studies completed to date show significant variability in the compositions, solid phase properties, and contaminant release characteristics from these residual tank wastes. This variability is the result of differences in waste chemistry/composition of wastes produced from several different spent fuel reprocessing schemes, subsequent waste reprocessing to remove certain target constituents, tank farm operations that concentrated wastes and mixed wastes between tanks, and differences in retrieval processes used to remove the wastes from the tanks. Release models were developed based upon results of chemical characterization of the bulk residual waste, solid-phase characterization (see companion paper 9277 by Krupka et al.), leaching and extraction experiments, and geochemical modeling. In most cases empirical release models were required to describe contaminant release from these wastes. Release of contaminants from residual waste was frequently found to be controlled by the solubility of phases that could not be identified and/or for which thermodynamic data and/or dissolution rates have not been measured. For example, significant fractions of Tc-99, I-129, and Cr appear to be coprecipitated at trace concentrations in metal oxide phases that could not be identified unambiguously. In the case of U release from tank 241-C-103 residual waste, geochemical calculations indicated that leachate

Radiological dose assessment for residual radioactive material in soil at the clean slate sites 1, 2, and 3, Tonopah Test Range

International Nuclear Information System (INIS)

1997-06-01

A radiological dose assessment has been performed for Clean Slate Sites 1, 2, and 3 at the Tonopah Test Range, approximately 390 kilometers (240 miles) northwest of Las Vegas, Nevada. The assessment demonstrated that the calculated dose to hypothetical individuals who may reside or work on the Clean Slate sites, subsequent to remediation, does not exceed the limits established by the US Department of Energy for protection of members of the public and the environment. The sites became contaminated as a result of Project Roller Coaster experiments conducted in 1963 in support of the US Atomic Energy Commission (Shreve, 1964). Remediation of Clean Slate Sites 1, 2, and 3 is being performed to ensure that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works on a Clean Slate site should not exceed 100 millirems per year. The DOE residual radioactive material guideline (RESRAD) computer code was used to assess the dose. RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines (Yu et al., 1993a). In May and June of 1963, experiments were conducted at Clean Slate Sites 1, 2, and 3 to study the effectiveness of earth-covered structures for reducing the dispersion of nuclear weapons material as a result of nonnuclear explosions. The experiments required the detonation of various simulated weapons using conventional chemical explosives (Shreve, 1964). The residual radioactive contamination in the surface soil consists of weapons grade plutonium, depleted uranium, and their radioactive decay products

Radiological dose assessment for residual radioactive material in soil at the clean slate sites 1, 2, and 3, Tonopah Test Range

Energy Technology Data Exchange (ETDEWEB)

NONE

1997-06-01

A radiological dose assessment has been performed for Clean Slate Sites 1, 2, and 3 at the Tonopah Test Range, approximately 390 kilometers (240 miles) northwest of Las Vegas, Nevada. The assessment demonstrated that the calculated dose to hypothetical individuals who may reside or work on the Clean Slate sites, subsequent to remediation, does not exceed the limits established by the US Department of Energy for protection of members of the public and the environment. The sites became contaminated as a result of Project Roller Coaster experiments conducted in 1963 in support of the US Atomic Energy Commission (Shreve, 1964). Remediation of Clean Slate Sites 1, 2, and 3 is being performed to ensure that the 50-year committed effective dose equivalent to a hypothetical individual who lives or works on a Clean Slate site should not exceed 100 millirems per year. The DOE residual radioactive material guideline (RESRAD) computer code was used to assess the dose. RESRAD implements the methodology described in the DOE manual for establishing residual radioactive material guidelines (Yu et al., 1993a). In May and June of 1963, experiments were conducted at Clean Slate Sites 1, 2, and 3 to study the effectiveness of earth-covered structures for reducing the dispersion of nuclear weapons material as a result of nonnuclear explosions. The experiments required the detonation of various simulated weapons using conventional chemical explosives (Shreve, 1964). The residual radioactive contamination in the surface soil consists of weapons grade plutonium, depleted uranium, and their radioactive decay products.

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

Directory of Open Access Journals (Sweden)

Mykuliak V. V.

2014-03-01

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

Antihypertensive Activity of Residue From “Gebto Arekei”. Locally ...

African Journals Online (AJOL)

Antihypertensive Activity of Residue From “Gebto Arekei”. Locally Distilled Medicinal Spirit From a Brew Containing Lupinus albus Seeds in Renovascular Hypertensive Guines-Pigs. Cherinet Ambaye, Tesfaye Tolessa, Abebe Abera, Hassen Taha Sherief, Dawit Abebe, Kelbessa Urga ...

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

International Nuclear Information System (INIS)

1978-10-01

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

Calculation of vibrational shifts of nitrile probes in the active site of ketosteroid isomerase upon ligand binding.

Science.gov (United States)

Layfield, Joshua P; Hammes-Schiffer, Sharon

2013-01-16

The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analogue equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active-site water molecule that is directly hydrogen-bonded to the nitrile probe, resulting in a more linear C≡N--H angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis.

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

International Nuclear Information System (INIS)

Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi; Takenaka, Hitoshi; Hamada, Minoru; Kuby, S.A.

1990-01-01

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP 2- and for AMP 2- in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP 2- and MgATP 2- . With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K m,app values for AMP 2- of the mutant enzymes, the relatively small increases in the K m,app values for MgATP 2- , and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

Science.gov (United States)

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Analysis of the technical capabilities of DOE sites for disposal of residuals from the treatment of mixed low-level waste

International Nuclear Information System (INIS)

Waters, R.D.; Gruebel, M.M.; Langkopf, B.S.; Kuehne, P.B.

1997-04-01

The US Department of Energy (DOE) has stored or expects to generate over the next five years more than 130,000 m 3 of mixed low-level waste (MLLW). Before disposal, MLLW is usually treated to comply with the land disposal restrictions of the Resource Conservation and Recovery Act. Depending on the type of treatment, the original volume of MLLW and the radionuclide concentrations in the waste streams may change. These changes must be taken into account in determining the necessary disposal capacity at a site. Treatment may remove the characteristic in some waste that caused it to be classified as mixed. Treatment of some waste may, by reduction of the mass, increase the concentrations of some transuranic radionuclides sufficiently so that it becomes transuranic waste. In this report, the DOE MLLW streams were analyzed to determine after-treatment volumes and radionuclide concentrations. The waste streams were reclassified as residual MLLW or low-level or transuranic waste resulting from treatment. The volume analysis indicated that about 89,000 m 3 of waste will require disposal as residual MLLW. Fifteen DOE sites were then evaluated to determine their capabilities for hosting disposal facilities for some or all of the residual MLLW. Waste streams associated with about 90% of the total residual MLLW volume are likely to present no significant issues for disposal and require little additional analysis. Future studies should focus on the remaining waste streams that are potentially problematic by examining site-specific waste acceptance criteria, alternative treatment processes, alternative waste forms for disposal, and pending changes in regulatory requirements

Release behavior of triazine residues in stabilised contaminated soils

International Nuclear Information System (INIS)

Ying, G.G.; Kookana, R.S.; Mallavarpu, M.

2005-01-01

This paper reports the release behavior of two triazines (atrazine and simazine) in stabilised soils from a pesticide-contaminated site in South Australia. The soils were contaminated with a range of pesticides, especially with triazine herbicides. With multiple extractions of each soil sample with deionised water (eight in total), 15% of atrazine and 4% of simazine residues were recovered, resulting in very high concentrations of the two herbicides in leachate. The presence of small fractions of surfactants was found to further enhance the release of the residues. Methanol content up to 10% did not substantially influence the concentration of simazine and atrazine released. The study demonstrated that while the stabilisation of contaminated soil with particulate activated carbon (5%) and cement mix (15%) was effective in locking the residues of some pesticides, it failed to immobilise triazine herbicides residues completely. Given the higher water solubility of these herbicides than other compounds more effective strategies to immobilise their residues is needed. - Stabilisation of contaminated soil with a mix of activated carbon and cement may fail to immobilise some contaminants like triazines

Long-term management of the existing radioactive wastes and residues at the Niagara Falls Storage Site. Draft Environmental Impact Statement

International Nuclear Information System (INIS)

1984-08-01

The statement assesses and compares several alternatives for long-term management of the existing radioactive wastes and residues at the Niagara Falls Storage Site (NFSS), Lewiston, New York. The alternatives include: (1) no action (continued interim storage at NFSS within a diked and capped containment area), (2) long-term management at NFSS (improved containment, with or without modified form of the residues), (3) long-term management at other DOE sites (Hanford, Washington, or Oak Ridge, Tennessee), and (4) offsite management of the residues at Hanford or Oak Ridge and either leaving the wastes at NFSS or removing them for disposal in the ocean. In addition to alternatives analyzed in depth, several options are also considered, including: other modifications of residue form, modification of the basic conceptual designs, other containment design options, transportation routes, and transportation modes. The radiological health effects (primarily increased risk of cancer) associated with long-term management of the wastes and residues are expected to be smaller than the nonradiological risks of occupational and transportation-related injuries and deaths. During the action period, the risk is highest for workers if all wastes and residues are moved to Hanford. The risk is highest for the general public if the residues are moved to Hanford and the wastes are moved to the ocean. Dispersal of the slightly contaminated wastes in the ocean is not expected to result in any significant impacts on the ocean environment or pose any significant radiological risk to humans. For all alternatives, if controls ceased, there would be eventual dispersion of the radioactive materials to the environment. If it is assumed that all controls cease, predicted time for loss of covers over the buried materials ranges from several hundred years to more than two million years, depending on the use of the land surface

A C. elegans-based foam for rapid on-site detection of residual live virus.

Energy Technology Data Exchange (ETDEWEB)

Negrete, Oscar A.; Branda, Catherine; Hardesty, Jasper O. E. (Sandia National Laboratories, Albuquerque, NM); Tucker, Mark David (Sandia National Laboratories, Albuquerque, NM); Kaiser, Julia N. (Global Product Management, Hilden, Germany); Kozina, Carol L.; Chirica, Gabriela S.

2012-02-01

In the response to and recovery from a critical homeland security event involving deliberate or accidental release of biological agents, initial decontamination efforts are necessarily followed by tests for the presence of residual live virus or bacteria. Such 'clearance sampling' should be rapid and accurate, to inform decision makers as they take appropriate action to ensure the safety of the public and of operational personnel. However, the current protocol for clearance sampling is extremely time-intensive and costly, and requires significant amounts of laboratory space and capacity. Detection of residual live virus is particularly problematic and time-consuming, as it requires evaluation of replication potential within a eukaryotic host such as chicken embryos. The intention of this project was to develop a new method for clearance sampling, by leveraging Sandia's expertise in the biological and material sciences in order to create a C. elegans-based foam that could be applied directly to the entire contaminated area for quick and accurate detection of any and all residual live virus by means of a fluorescent signal. Such a novel technology for rapid, on-site detection of live virus would greatly interest the DHS, DoD, and EPA, and hold broad commercial potential, especially with regard to the transportation industry.

Computer Simulations Reveal Multiple Functions for Aromatic Residues in Cellulase Enzymes (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-07-01

NREL researchers use high-performance computing to demonstrate fundamental roles of aromatic residues in cellulase enzyme tunnels. National Renewable Energy Laboratory (NREL) computer simulations of a key industrial enzyme, the Trichoderma reesei Family 6 cellulase (Cel6A), predict that aromatic residues near the enzyme's active site and at the entrance and exit tunnel perform different functions in substrate binding and catalysis, depending on their location in the enzyme. These results suggest that nature employs aromatic-carbohydrate interactions with a wide variety of binding affinities for diverse functions. Outcomes also suggest that protein engineering strategies in which mutations are made around the binding sites may require tailoring specific to the enzyme family. Cellulase enzymes ubiquitously exhibit tunnels or clefts lined with aromatic residues for processing carbohydrate polymers to monomers, but the molecular-level role of these aromatic residues remains unknown. In silico mutation of the aromatic residues near the catalytic site of Cel6A has little impact on the binding affinity, but simulation suggests that these residues play a major role in the glucopyranose ring distortion necessary for cleaving glycosidic bonds to produce fermentable sugars. Removal of aromatic residues at the entrance and exit of the cellulase tunnel, however, dramatically impacts the binding affinity. This suggests that these residues play a role in acquiring cellulose chains from the cellulose crystal and stabilizing the reaction product, respectively. These results illustrate that the role of aromatic-carbohydrate interactions varies dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, the results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering

Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

Science.gov (United States)

Inaba, Juliana; Thornton, Jeremy; Huergo, Luciano Fernandes; Monteiro, Rose Adele; Klassen, Giseli; Pedrosa, Fábio de Oliveira; Merrick, Mike; de Souza, Emanuel Maltempi

2015-02-01

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP. Copyright © 2014 Elsevier GmbH. All rights reserved.

Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

International Nuclear Information System (INIS)

Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

2010-01-01

Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

Identification of Residues Important for the Activity of Haloferax volcanii AglD, a Component of the Archaeal N-Glycosylation Pathway

Directory of Open Access Journals (Sweden)

Lina Kaminski

2010-01-01

Full Text Available In Haloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in an Hfx. volcanii aglD deletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

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

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Haibo Yu

2007-02-01

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

Vision restoration after brain and retina damage: the "residual vision activation theory".

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Sabel, Bernhard A; Henrich-Noack, Petra; Fedorov, Anton; Gall, Carolin

2011-01-01

Vision loss after retinal or cerebral visual injury (CVI) was long considered to be irreversible. However, there is considerable potential for vision restoration and recovery even in adulthood. Here, we propose the "residual vision activation theory" of how visual functions can be reactivated and restored. CVI is usually not complete, but some structures are typically spared by the damage. They include (i) areas of partial damage at the visual field border, (ii) "islands" of surviving tissue inside the blind field, (iii) extrastriate pathways unaffected by the damage, and (iv) downstream, higher-level neuronal networks. However, residual structures have a triple handicap to be fully functional: (i) fewer neurons, (ii) lack of sufficient attentional resources because of the dominant intact hemisphere caused by excitation/inhibition dysbalance, and (iii) disturbance in their temporal processing. Because of this resulting activation loss, residual structures are unable to contribute much to everyday vision, and their "non-use" further impairs synaptic strength. However, residual structures can be reactivated by engaging them in repetitive stimulation by different means: (i) visual experience, (ii) visual training, or (iii) noninvasive electrical brain current stimulation. These methods lead to strengthening of synaptic transmission and synchronization of partially damaged structures (within-systems plasticity) and downstream neuronal networks (network plasticity). Just as in normal perceptual learning, synaptic plasticity can improve vision and lead to vision restoration. This can be induced at any time after the lesion, at all ages and in all types of visual field impairments after retinal or brain damage (stroke, neurotrauma, glaucoma, amblyopia, age-related macular degeneration). If and to what extent vision restoration can be achieved is a function of the amount of residual tissue and its activation state. However, sustained improvements require repetitive

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

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Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

2007-10-01

Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

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Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

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Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

2018-02-12

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

Mapping the active site helix-to-strand conversion of CxxxxC peroxiredoxin Q enzymes.

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Perkins, Arden; Gretes, Michael C; Nelson, Kimberly J; Poole, Leslie B; Karplus, P Andrew

2012-09-25

Peroxiredoxins (Prx) make up a family of enzymes that reduce peroxides using a peroxidatic cysteine residue; among these, members of the PrxQ subfamily are proposed to be the most ancestral-like yet are among the least characterized. In many PrxQ enzymes, a second "resolving" cysteine is located five residues downstream from the peroxidatic Cys, and these residues form a disulfide during the catalytic cycle. Here, we describe three hyperthermophilic PrxQ crystal structures originally determined by the RIKEN structural genomics group. We reprocessed the diffraction data and conducted further refinement to yield models with R(free) values lowered by 2.3-7.2% and resolution extended by 0.2-0.3 Å, making one, at 1.4 Å, one of the best resolved peroxiredoxins to date. Comparisons of two matched thiol and disulfide forms reveal that the active site conformational change required for disulfide formation involves a transition of ~20 residues from a pair of α-helices to a β-hairpin and 3(10)-helix. Each conformation has ~10 residues with a high level of disorder providing slack that allows the dramatic shift, and the two conformations are anchored to the protein core by distinct nonpolar side chains that fill three hydrophobic pockets. Sequence conservation patterns confirm the importance of these and a few additional residues for function. From a broader perspective, this study raises the provocative question of how to make use of the valuable information in the Protein Data Bank generated by structural genomics projects but not described in the literature, perhaps remaining unrecognized and certainly underutilized.

Solid radioactive waste: evaluation of residual activity in nuclear medicine services

International Nuclear Information System (INIS)

Alabarse, Frederico G.; Xavier, Ana M.; Magalhaes, Maisa H.; Guerrero, Jesus S.P.

2009-01-01

An experimental programme to estimate, with a better degree of accuracy, the activity that remains adsorbed in flasks and syringes used in Nuclear Medicine Services for the administration of radionuclides to patients submitted to diagnostic or therapy is been conducted under the coordination of the Radioactive Waste Division of the Brazilian Nuclear Energy Commission, CNEN. The adopted recommendation in Brazil to allow an expedite solid waste management in nuclear medicine facilities, up to the present, is to consider that 2% of the initial activity remains adsorbed in the solid waste, which easily allows the calculation of the storage time to achieve regulatory clearance levels by decay. This research evaluates 17 different kinds of radiopharmaceuticals and three radioisotopes: 99m Tc, 67 Ga and 201 Tl. Results obtained by means of a weighting method to estimate the residual mass in flasks show that the ratio of the mass of the liquid that remains in the solid waste to the mass of the empty flask is constant. This suggests that the residual activity depends on the initial activity concentration of radiopharmaceutical contained in each flask, as assumed by the regulatory body. Additionally, results obtained by determining the remaining activity in flasks, shortly after the injection of its radionuclide contents in patients, indicate that an average value for the residual activity of the order of 10% of the initial activity contained in the flasks or syringes should be adopted to determine the decay storage time before the release of solid waste in the urban conventional land fill disposal system. The 'rule of thumb' of 10 half-lives for storage before clearance is also discussed in the present work. (author)

Structural and Functional Consequences of Chaperone Site Deletion in αA-Crystallin

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Santhoshkumar, Puttur; Karmakar, Srabani; Sharma, Krishna K.

2016-01-01

The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70–88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70–76 and αAΔ70–88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70–88 was completely inactive as a chaperone, while αAΔ70–76 demonstrated a 40–50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70–88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45 °C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70–88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53 °C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70–88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70–88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins. PMID:27524665

The arginine residue within the C-terminal active core of Bombyx mori pheromone biosynthesis-activating neuropeptide (PBAN is essential for receptor binding and activation

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Takeshi eKawai

2012-03-01

Full Text Available In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis activating neuropeptide (PBAN. Bombyx mori PBAN (BomPBAN consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R residue two positions from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to reveal the role of the Arg residue in the BomPBAN active core. We synthesized a ten-residue peptide corresponding to the C-terminal part of BomPBAN with a series of point mutants at the 2nd position (ie, Arg from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells concomitantly expressing a fluorescent PBAN receptor chimera (PBANR-EGFP and loaded with the fluorescent Ca2+ indicator, Fura Red-AM. PBAN analogs with the C2 position replaced with alanine (Ala, A, aspartic acid (Asp, D, serine (Ser, S or L-2-aminooctanoic acid (Aoc decreased PBAN-like activity. RC2A (SKTRYFSPALamide and RC2D (SKTRYFSPDLamide had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide. We also prepared Rhodamine Red-labeled PBAN analogs of the mutants and examined their ability to bind PBANR. In contrast to 100 nM Rhodamine Red-PBAN C10, none of the mutants at the same concentration exhibited PBANR binding. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation.

Computation Of The Residual Radionuclide Activity Within Three Natural Waterways At The Savannah River Site

Energy Technology Data Exchange (ETDEWEB)

Hiergesell, R. A.; Phifer, M. A.

2014-01-07

In 2010 a Composite Analysis (CA) of the U.S. Department of Energy’s (DOE’s) Savannah River Site (SRS) was completed. This investigation evaluated the dose impact of the anticipated SRS End State residual sources of radionuclides to offsite members of the public. Doses were assessed at the locations where SRS site streams discharge into the Savannah River at the perimeter of the SRS. Although the model developed to perform this computation indicated that the dose constraint of 0.3 mSv/yr (30 mrem/yr), associated with CA, was not approached at the Points of Assessment (POAs), a significant contribution to the total computed dose was derived from the radionuclides (primarily Cs-137) bound-up in the soil and sediment of the drainage corridors of several SRS streams. DOE’s Low Level Waste Federal Review Group (LFRG) reviewed the 2010 CA and identified several items to be addressed in the SRS Maintenance Program. One of the items recognized Cs-137 in the Lower Three Runs (LTR) Integrator Operable Unit (IOU), as a significant CA dose driver. The item made the recommendation that SRS update the estimated radionuclide inventory, including Cs-137, in the LTR IOU. That initial work has been completed and its radionuclide inventory refined. There are five additional streams at SRS and the next phase of the response to the LFRG concern was to obtain a more accurate inventory and distribution of radionuclides in three of those streams, Fourmile Branch (FMB), Pen Branch (PB) and Steel Creek (SC). Each of these streams is designated as an IOU, which are defined for the purpose of this investigation as the surface water bodies and associated wetlands, including the channel sediment, floodplain sed/soil, and related biota. If present, radionuclides associated with IOUs are adsorbed to the streambed sediment and soils of the shallow floodplains that lie immediately adjacent to stream channels. The scope of this effort included the evaluation of any previous sampling and

A tiered analytical protocol for the characterization of heavy oil residues at petroleum-contaminated hazardous waste sites

International Nuclear Information System (INIS)

Pollard, S.J.T.; Kenefick, S.L.; Hrudey, S.E.; Fuhr, B.J.; Holloway, L.R.; Rawluk, M.

1994-01-01

The analysis of hydrocarbon-contaminated soils from abandoned refinery sites in Alberta, Canada is used to illustrate a tiered analytical approach to the characterization of complex hydrocarbon wastes. Soil extracts isolated from heavy oil- and creosote-contaminated sites were characterized by thin layer chromatography with flame ionization detection (TLC-FID), ultraviolet fluorescence, simulated distillation (GC-SIMDIS) and chemical ionization GC-MS analysis. The combined screening and detailed analytical methods provided information essential to remedial technology selection including the extent of contamination, the class composition of soil extracts, the distillation profile of component classes and the distribution of individual class components within various waste fractions. Residual contamination was characteristic of heavy, degraded oils, consistent with documented site operations and length of hydrocarbon exposure at the soil surface

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD.

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Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping

2017-06-19

Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light-dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

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Joel Raborn

Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

Radioactive residues at nuclear sites in the United States of America

International Nuclear Information System (INIS)

Fiore, J.; Rampertaap, A.; Greeves, J.; MacKinney, J.; Raguso, M.; Selstrom, J.

2000-01-01

The United States of America has a large number of sites where radioactive materials have been mined, processed, produced, or used. Upon completion of activities at these sites, some will be cleaned up completely and released for unrestricted future use. However, at other sites, contamination will remain on the site following cleanup, requiring long term institutional controls. Depending on the purpose or the activity conducted at a specific site, these locations are generally under the jurisdiction of a federal agency: the United States Department of Energy, the United States Nuclear Regulatory Commission, the United States Environmental Protection Agency, or the United States Department of Defense. However, under certain conditions, the Nuclear Regulatory Commission relinquishes regulatory authority to individual states to regulate the commercial uses of radioactive materials (except for nuclear reactors), so some locations with radioactive materials are under the jurisdiction of individual states. Other sites with naturally occurring radioactive contamination may also be controlled by individual states. One or two sites under the jurisdiction of each agency are discussed where radioactive materials either existed in the past or exist today, and the source of contamination, the anticipated end state, and the process by which the responsible agency did or will conduct cleanup and site closure are described. Several issues are reviewed that must be addressed in order to design and implement remediations that will ensure long term protection for the environment and future inhabitants. The role of citizens in environmental cleanups is examined, and how institutional controls may be applied to ensure long term protection of remediations that leave some contaminants in place. (author)

A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

Science.gov (United States)

The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

Evidence of superstoichiometric H/D LENR active sites and high-temperature superconductivity in a hydrogen-cycled Pd/PdO

International Nuclear Information System (INIS)

Lipson, A.G.; Castano, C.H.; Miley, G.H.; Lyakhov, B.F.; Tsivadze, A.Yu.; Mitin, A.V.

2006-01-01

Electron transport and magnetic properties have been studied in a 12.5 μm thick Pd foil with a thermally grown oxide and a low-residual concentration of hydrogen. This foil was deformed by cycling across the Pd hydride miscibility gap and the residual hydrogen was trapped at dislocation cores. Anomalies of both resistance and magnetic susceptibility have been observed below 70 K, indicating the appearance of excess conductivity and a diamagnetic response that we interpret in terms of filamentary superconductivity. These anomalies are attributed to a condensed hydrogen-rich phase at dislocation cores. The role of deuterium rich dislocation cores as LENR active sites is discussed. (author)

Evidence of Superstoichiometric H/d Lenr Active Sites and High-Temperature Superconductivity in a Hydrogen-Cycled Pd/PdO

Science.gov (United States)

Lipson, A. G.; Castano, C. H.; Miley, G. H.; Lyakhov, B. F.; Tsivadze, A. Yu.; Mitin, A. V.

Electron transport and magnetic properties have been studied in a 12.5 μm thick Pd foil with a thermally grown oxide and a low-residual concentration of hydrogen. This foil was deformed by cycling across the Pd hydride miscibility gap and the residual hydrogen was trapped at dislocation cores. Anomalies of both resistance and magnetic susceptibility have been observed below 70 K, indicating the appearance of excess conductivity and a diamagnetic response that we interpret in terms of filamentary superconductivity. These anomalies are attributed to a condensed hydrogen-rich phase at dislocation cores. The role of deuterium rich dislocation cores as LENR active sites is discussed.

Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

Science.gov (United States)

Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

2003-02-01

We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

Correlation of beam loss to residual activation in the AGS

International Nuclear Information System (INIS)

Brown, K.A.

1991-01-01

Studies of beam loss and activation at the AGS have provided a better understanding of measurements of beam loss and how they may be used to predict activation. Studies have been done in which first order correlations have been made between measured beam losses on the distributed ionization chamber system in the AGS and the health physics recorded residual activation. These studies have provided important insight into the ionization chamber system, its limitations, and its usefulness in the prediction of activation based on monitored beam loss. In recent years the AGS has run high intensity protons primarily for rare kaon decay experiments. In this mode of running the AGS typically accelerates beam from an injection momentum of 0.644 GeV/c up to a slow extracted beam (SEB) momentum of 24.2 GeV/c. The beam intensities are on the order of 4.5 x 10 13 protons per AGS cycle at injection to as high as 1.9 x 10 13 protons per AGS cycle at extraction. Residual activation varies around the AGS ring from the order of 5 mR/hour to levels of the order at 5 R/hour. The highest levels occur around the AGS beam catcher and the extraction equipment

Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

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Patrick E F O'Rourke

Full Text Available 4-Amino-4-deoxychorismate lyase (PabC catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5'-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp(2 to sp(3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of

Mapping the Active Site Helix-to-Strand Conversion of CxxxxC Peroxiredoxin Q Enzymes †

Science.gov (United States)

Perkins, Arden; Gretes, Michael C.; Nelson, Kimberly J.; Poole, Leslie B.; Karplus, P. Andrew

2012-01-01

Peroxiredoxins (Prx) are a family of enzymes which reduce peroxides using a peroxidatic cysteine residue; among these, the PrxQ subfamily members are proposed to be the most ancestral-like yet are among the least characterized. In many PrxQ enzymes, a second “resolving” cysteine is located six residues downstream from the peroxidatic Cys, and these residues form a disulfide during the catalytic cycle. Here, we describe three hyperthermophilic PrxQ crystal structures originally solved by the RIKEN structural genomics group. We reprocessed the diffraction data and carried out further refinement to yield models with Rfree lowered by 2.3–7.2% and resolution extended by 0.2–0.3 Å, making one, at 1.4 Å, the best resolved peroxiredoxin to date. Comparisons of two matched thiol and disulfide forms reveal that the active site conformational change required for disulfide formation involves a transition of about 20 residues from a pair of α-helices to a β-hairpin and 310-helix. Each conformation has about 10 residues with high disorder providing slack that enables the dramatic shift, and the two conformations are anchored to the protein core by distinct non-polar side chains that fill three hydrophobic pockets. Sequence conservation patterns confirm the importance of these and a few additional residues for function. From a broader perspective, this study raises the provocative question of how to make use of the valuable information in the protein data bank generated by structural genomics projects but not described in the literature, perhaps remaining unrecognized and certainly underutilized. PMID:22928725

TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues

OpenAIRE

Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

2014-01-01

microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents ...

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

Science.gov (United States)

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

Residual radioactivity in the vicinity of formerly utilized MED/AEC sites

International Nuclear Information System (INIS)

Haywood, F.F.; Goldsmith, W.A.

1980-01-01

As demand for uranium and thorium was accelerated during the 1940's, services of chemical and metallurgical firms and major research facilities were contracted as needed by the Manhattan Engineer District (MED). A lack of documentation of the radiological status at the time contracts were terminated at these facilities led the Department of Energy (DOE), and its predecessor the Energy Research and Development Administration (ERDA), to develop a major radiological resurvey program to fill this information void. A combination of aerial and ground-level radiological monitoring teams were utilized to identify and assess off-site radioactivity. Results from comprehensive aerial surveys provide the approximate areal extent of elevated radiation levels on the ground. These aerial survey results led to two types of ground-level surveys: (1) gamma-ray scanning on foot or from a motorized vehicle (mobile lab based system) to pinpoint the location of residual radioactivity; and (2) compehensive radiological surveys to determine the amount and type of materials present on specific parcels of private and public property identified during the scanning. This type of investigation was initiated in 1978 and has been successful in identifying and assessing the potential radiation hazard from property on which materials bearing natural radioactivity have been found. This paper contains a description of the techniques used to find and evaluate radioactive material displaced outside the boundaries of a formerly utilized site

Mutational analysis to identify the residues essential for the inhibition of N-acetyl glutamate kinase of Corynebacterium glutamicum.

Science.gov (United States)

Huang, Yuanyuan; Zhang, Hao; Tian, Hongming; Li, Cheng; Han, Shuangyan; Lin, Ying; Zheng, Suiping

2015-09-01

N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine that is inhibited by its end product L-arginine in Corynebacterium glutamicum (C. glutamicum). In this study, the potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling, inhibitor docking, and site-directed mutagenesis. The allosteric inhibition of NAGK was successfully alleviated by a mutation, as determined through analysis of mutant enzymes, which were overexpressed in vivo in C. glutamicum ATCC14067. Analysis of the mutant enzymes and docking analysis demonstrated that residue W23 positions an arginine molecule, and the interaction between arginine and residues L282, L283, and T284 may play an important role in the remote inhibitory process. Based on the results of the docking analysis of the effective mutants, we propose a linkage mechanism for the remote allosteric regulation of NAGK activity, in which residue R209 may play an essential role. In this study, the structure of the arginine-binding site of C. glutamicum NAGK (CgNAGK) was successfully predicted and the roles of the relevant residues were identified, providing new insight into the allosteric regulation of CgNAGK activity and a solid platform for the future construction of an optimized L-arginine producing strain.

Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

International Nuclear Information System (INIS)

Ruel, Nancy; Zago, Anna; Spear, Patricia G.

2006-01-01

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity

Neutron activation analysis for noble metals in matte leach residues

International Nuclear Information System (INIS)

Hart, R.J.

1978-01-01

The development of the neutron activation analysis technique as a method for rapid and precise determinations of platinum group metals in matte leach residues depends on obtaining a method for effecting complete and homogeneous sample dilution. A simple method for solid dilution of metal samples is outlined in this study, which provided a basis for the accurate determination of all the noble metals by the Neutron Activation Analysis technique

Application of activated carbons from coal and coconut shell for removing free residual chlorine.

Science.gov (United States)

Ogata, Fumihiko; Tominaga, Hisato; Ueda, Ayaka; Tanaka, Yuko; Iwata, Yuka; Kawasaki, Naohito

2013-01-01

This study investigated the removal of free residual chlorine by activated carbon (AC). ACs were prepared from coal (AC1) and coconut shell (AC2). The specific surface area of AC1 was larger than that of AC2. The removal of free residual chlorine increased with elapsed time and amount of adsorbent. The removal mechanism of free residual chlorine was the dechlorination reaction between hypochlorous acid or hypochlorite ion and AC. Moreover, AC1 was useful in the removal of free residual chlorine in tap water. The optimum condition for the removal of free residual chlorine using a column is space velocity 306 1/h; liner velocity 6.1 m/h.

Reciprocally coupled residues crucial for protein kinase Pak2 activity calculated by statistical coupling analysis.

Directory of Open Access Journals (Sweden)

Yuan-Hao Hsu

2010-03-01

Full Text Available Regulation of Pak2 activity involves at least two mechanisms: (i phosphorylation of the conserved Thr(402 in the activation loop and (ii interaction of the autoinhibitory domain (AID with the catalytic domain. We collected 482 human protein kinase sequences from the kinome database and globally mapped the evolutionary interactions of the residues in the catalytic domain with Thr(402 by sequence-based statistical coupling analysis (SCA. Perturbation of Thr(402 (34.6% suggests a communication pathway between Thr(402 in the activation loop, and Phe(387 (DeltaDeltaE(387F,402T = 2.80 in the magnesium positioning loop, Trp(427 (DeltaDeltaE(427W,402T = 3.12 in the F-helix, and Val(404 (DeltaDeltaE(404V,402T = 4.43 and Gly(405 (DeltaDeltaE(405G,402T = 2.95 in the peptide positioning loop. When compared to the cAMP-dependent protein kinase (PKA and Src, the perturbation pattern of threonine phosphorylation in the activation loop of Pak2 is similar to that of PKA, and different from the tyrosine phosphorylation pattern of Src. Reciprocal coupling analysis by SCA showed the residues perturbed by Thr(402 and the reciprocal coupling pairs formed a network centered at Trp(427 in the F-helix. Nine pairs of reciprocal coupling residues crucial for enzymatic activity and structural stabilization were identified. Pak2, PKA and Src share four pairs. Reciprocal coupling residues exposed to the solvent line up as an activation groove. This is the inhibitor (PKI binding region in PKA and the activation groove for Pak2. This indicates these evolutionary conserved residues are crucial for the catalytic activity of PKA and Pak2.

A manual for implementing residual radioactive material guidelines

International Nuclear Information System (INIS)

Gilbert, T.L.; Yu, C.; Yuan, Y.C.; Zielen, A.J.; Jusko, M.J.; Wallo, A. III; Argonne National Lab., IL; Dames and Moore, West Valley, NY; Argonne National Lab., IL; USDOE Assistant Secretary for Nuclear Energy, Washington, DC

1989-06-01

This manual presents information for implementing US Department of Energy (DOE) guidelines for residual radioactive material at sites identified by the Formerly Utilized Sites Remedial Action Program (FUSRAP) and the Surplus Facilities Management Program (SFMP). It describes the analysis and models used to derive site-specific guidelines for allowable residual concentrations of radionuclides in soil and the design and use of the RESRAD computer code for calculating guideline values. It also describes procedures for implementing DOE policy for reducing residual radioactivity to levels that are as low as reasonably achievable. 36 refs., 16 figs, 22 tabs

Remaining Sites Verification Package for the 128-B-3 Burn Pit Site, Waste Site Reclassification Form 2006-058

Energy Technology Data Exchange (ETDEWEB)

L. M. Dittmer

2006-11-17

The 128-B-3 waste site is a former burn and disposal site for the 100-B/C Area, located adjacent to the Columbia River. The 128-B-3 waste site has been remediated to meet the remedial action objectives specified in the Remaining Sites ROD. The results of verification sampling demonstrated that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results of sampling at upland areas of the site also showed that residual contaminant concentrations are protective of groundwater and the Columbia River.

Activated carbon from flash pyrolysis of eucalyptus residue

Directory of Open Access Journals (Sweden)

Grima-Olmedo C

2016-09-01

Full Text Available Forestry waste (eucalyptus sp was converted into activated carbon by initial flash pyrolysis followed carbonization and CO2 activation. These residues were obtained from a pilot plant in Spain that produces biofuel, the biochar represented 10–15% in weight. It was observed that the highest activation was achieved at a temperature of 800 °C, the specific surface increased with time but, on the contrary, high loss of matter was observed. At 600 °C, although there was an important increase of the specific surface and the volume of micropores, at this temperature it was observed that the activation time was not an influential parameter. Finally, at 400 °C it was observed that the activation process was not very significant. Assessing the average pore diameter it was found that the lowest value corresponded to the activation temperature of 600 °C, which indicated the development of microporosity. When the activation temperature increases up to 800 °C the pore diameter increased developing mesoporosity.

In situ chemical composition measurement of individual cloud residue particles at a mountain site, southern China

Directory of Open Access Journals (Sweden)

Q. Lin

2017-07-01

Full Text Available To investigate how atmospheric aerosol particles interact with chemical composition of cloud droplets, a ground-based counterflow virtual impactor (GCVI coupled with a real-time single-particle aerosol mass spectrometer (SPAMS was used to assess the chemical composition and mixing state of individual cloud residue particles in the Nanling Mountains (1690 m a. s. l. , southern China, in January 2016. The cloud residues were classified into nine particle types: aged elemental carbon (EC, potassium-rich (K-rich, amine, dust, Pb, Fe, organic carbon (OC, sodium-rich (Na-rich and Other. The largest fraction of the total cloud residues was the aged EC type (49.3 %, followed by the K-rich type (33.9 %. Abundant aged EC cloud residues that mixed internally with inorganic salts were found in air masses from northerly polluted areas. The number fraction (NF of the K-rich cloud residues increased within southwesterly air masses from fire activities in Southeast Asia. When air masses changed from northerly polluted areas to southwesterly ocean and livestock areas, the amine particles increased from 0.2 to 15.1 % of the total cloud residues. The dust, Fe, Pb, Na-rich and OC particle types had a low contribution (0.5–4.1 % to the total cloud residues. Higher fraction of nitrate (88–89 % was found in the dust and Na-rich cloud residues relative to sulfate (41–42 % and ammonium (15–23 %. Higher intensity of nitrate was found in the cloud residues relative to the ambient particles. Compared with nonactivated particles, nitrate intensity decreased in all cloud residues except for dust type. To our knowledge, this study is the first report on in situ observation of the chemical composition and mixing state of individual cloud residue particles in China.

The C-terminal N-glycosylation sites of the human α1,3/4-fucosyltransferase III, -V and -VI (hFucTIII, -V and -VI) are necessary for the expression of full enzyme activity

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Jensen, Uffe Birk; Bross, Peter Gerd

2000-01-01

FucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced...

Pathways analysis and radiation-dose estimates for radioactive residues at formerly utilized MED/AEC sites

Energy Technology Data Exchange (ETDEWEB)

Gilbert, T.L.; Chee, P.C.; Knight, M.J.; Peterson, J.M.; Roberts, C.J.; Robinson, J.E.; Tsai, S.Y.H.; Yuan, Y.C.

1983-03-01

Methods of analysis are developed for estimating the largest individual radiation dose that could result from residual radioactivity at sites identified by the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the US Department of Energy. Two unique aspects of the methods are (1) a systematic structuring of the radiation pathways analysis into source terms, source-to-exposure analysis, and exposure-to-dose analysis, and (2) the systematic use of data on the average concentrations of naturally occurring radionuclides in soil, food, and the human body in order to assess the validity of model calculations and obtain more realistic values. The methods are applied to a typical FUSRAP site in order to obtain generic source-to-dose (D/S) conversion factors for estimating the radiation dose to the maximally exposed individual from a known concentration of radionuclides in the soil. The D/S factors are used to derive soil guidelines, i.e., the limiting concentrations of radionuclides at a typical FUSRAP site that are unlikely to result in individual dose limits that exceed generally accepted radiation protection standards. The results lead to the conclusion that the soil guidelines should not exceed 17, 75, and 300 pCi/g for Ra-226, U-238, and Th-230, respectively.

Pathways analysis and radiation-dose estimates for radioactive residues at formerly utilized MED/AEC sites

International Nuclear Information System (INIS)

Gilbert, T.L.; Chee, P.C.; Knight, M.J.; Peterson, J.M.; Roberts, C.J.; Robinson, J.E.; Tsai, S.Y.H.; Yuan, Y.C.

1983-03-01

Methods of analysis are developed for estimating the largest individual radiation dose that could result from residual radioactivity at sites identified by the Formerly Utilized Sites Remedial Action Program (FUSRAP) of the US Department of Energy. Two unique aspects of the methods are (1) a systematic structuring of the radiation pathways analysis into source terms, source-to-exposure analysis, and exposure-to-dose analysis, and (2) the systematic use of data on the average concentrations of naturally occurring radionuclides in soil, food, and the human body in order to assess the validity of model calculations and obtain more realistic values. The methods are applied to a typical FUSRAP site in order to obtain generic source-to-dose (D/S) conversion factors for estimating the radiation dose to the maximally exposed individual from a known concentration of radionuclides in the soil. The D/S factors are used to derive soil guidelines, i.e., the limiting concentrations of radionuclides at a typical FUSRAP site that are unlikely to result in individual dose limits that exceed generally accepted radiation protection standards. The results lead to the conclusion that the soil guidelines should not exceed 17, 75, and 300 pCi/g for Ra-226, U-238, and Th-230, respectively

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions.

Science.gov (United States)

Liang, Jing; Han, Qian; Ding, Haizhen; Li, Jianyong

2017-12-01

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO 2 , NH 3, and H 2 O 2 . This contrasts to the typical DDC-catalyzed reaction, which produces CO 2 and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H 2 O 2 in the process. Biochemical assessment established that H 2 O 2 , formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H 2

Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

Science.gov (United States)

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

2015-01-01

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

Enzymatic activity measured by microcalorimetry in soil amended with organic residues

Directory of Open Access Journals (Sweden)

Karina Cenciani

2011-08-01

Full Text Available Enzymatic activity is an important property for soil quality evaluation. Two sequences of experiments were carried out in order to evaluate the enzymatic activity in a soil (Rhodic Eutrudox amended with cattle manure, earthworm casts, or sewage sludges from the municipalities of Barueri and Franca. The activity of commercial enzymes was measured by microcalorimetry in the same soil samples after sterilization. In the first experiment, the enzyme activities of cellulase, protease, and urease were determined in the soil samples during a three month period. In the second sequence of experiments, the thermal effect of the commercial enzymes cellulase, protease, and urease on sterilized soil samples under the same tretaments was monitored for a period of 46 days. The experimental design was randomized and arranged as factorial scheme in five treatments x seven samplings with five replications. The treatment effects were statistically evaluated by one-way analysis of variance. Tukey´s test was used to compare means at p < 0.05. The presence of different sources of organic residues increased the enzymatic activity in the sampling period. Cattle manure induced the highest enzymatic activity, followed by municipal sewage sludge, whereas earthworm casts induced the lowest activity, but differed from control treatment. The thermal effect on the enzyme activity of commercial cellulase, protease, and urease showed a variety of time peaks. These values probably oscillated due to soil physical-chemical factors affecting the enzyme activity on the residues.

Impacts of Residual Surfactant on Tetrachloroethene (PCE) Degradation Following Pilot-Scale SEAR Treatment at a Chloroethene-Impacted Site

Science.gov (United States)

Ramsburg, C. A.; Abriola, L. M.; Pennell, K. D.; Löffler, F. E.; Gamache, M.; Petrovskis, E. A.

2003-04-01

A pilot-scale surfactant-enhanced aquifer remediation (SEAR) demonstration was completed during the summer of 2000 at the Bachman Road site (Oscoda, MI USA). For this test, an aqueous solution of 60 g/L Tween 80 (polyoxyethylene (20) sorbitan monooleate) was used to recover tetrachloroethene (PCE) from a suspected source zone, located underneath a former dry-cleaning facility. Tween 80 was selected for use based upon its demonstrated capacity to solubilize PCE, “food-grade” status, and biodegradative potential. Hydraulic control was maintained throughout the test, with 95% of the injected surfactant mass recovered by a single extraction well. Source-zone monitoring conducted 15 months after SEAR treatment revealed the presence of previously undetected volatile fatty acids (acetate and formate) and PCE degradation products (trichloroethene, cis-1,2-dichloroethene, trans-1,2-dichlorethene, and vinyl chloride), in conjunction with PCE concentration reductions of approximately two orders-of-magnitude. The detection of volatile fatty acids is relevant, as they are likely fermentation products of residual Tween 80. Microbial reductive dechlorination is limited by available electron donors, and microcosm studies demonstrated that both acetate and formate support reductively dechlorinating populations present at the oligotrophic Bachman Road site aquifer. Surfactant transport simulations, using a regional flow model developed for the site, were employed to determine appropriate down-gradient monitoring locations. Drive point samples taken 15 months post-treatment in the vicinity of the simulated residual surfactant plume, contained elevated concentrations of acetate and PCE daughter products. Ongoing efforts include continued site-monitoring, and microcosm studies to corroborate a causal relationship between Tween 80 fermentation and PCE dechlorination.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays

NARCIS (Netherlands)

M. Pieters (Marlien); S.A. Barnard (Sunelle A.); D.T. Loots (Du Toit); D.C. Rijken (Dingeman)

2017-01-01

textabstractDue to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen

Effect of fire residues (ash and char) on microbial activity, respiration and methanogenesis in three subtropical wetland soils

Science.gov (United States)

Medvedeff, C.; Hogue, B.; Inglett, P.

2011-12-01

Prescribed fire is a common restoration and maintenance technique in the southern United States. Prescribed burns coupled with frequent natural fires in South Florida can have devastating effects on ecosystem function. To determine the effect fire residues have on carbon biogeochemical cycling litter material was obtained from two restored and one native marl wetland in Everglades National Park and manipulated in a laboratory setting to produce ash and vegetation derived char. Based on vegetation biomass removal pre and post fire (insitu) appropriate aliquots of each fire residue was added to experimental microcosms as a soil amendment. Soil enzymes (β-glucosidase, cellobiohydrolase, phosphatase, bis-phosphate and leucine amino peptidase), aerobic and anaerobic respiration (CO2) potentials, extractable C and methanogenesis were measured over a 25 day period. Regardless of site C enzymes responded to both amendments within 5 days of addition. Similarly amended soil contained more extractable carbon in the reference and one of the restored sites. In the restored sites ash and char inhibited methanogenesis, had no effect on anaerobic CO2 potentials, but stimulated aerobic respiration after ten days. In contrast, within the first ten days phosphatase enzyme activity was lower in the ash treatment when compared to the control treatment and stimulation of aerobic respiration was observed in both treatment soils. After ten days ash stimulated methanogenic processing while suppressing anaerobic CO2 production suggesting methanogens in this ecosystem may be dependant on usable carbon substrates derived from aerobic microbial processing. This study illustrates the variable response of C parameters to complete and incomplete combusted materials produced from both prescribed and natural fires with particular importance to fire adapted ecosystems.

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship.

Science.gov (United States)

Wang, Qing; Bao, Luyao; Jia, Chenjun; Li, Mei; Li, Jian-Jun; Lu, Xuefeng

2017-03-16

Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k cat value below 1 min -1 , which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required. The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%. Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.

Efficient oxygen electrocatalysis on special active sites

DEFF Research Database (Denmark)

Halck, Niels Bendtsen

throughout this thesis to understand these local structure effects and their influence on surface reactions. The concept of these special active sites is used to explain how oxygen evolution reaction (OER) catalysts can have activities beyond the limits of what was previously thought possible. The concept...... stored in these bonds in an eco-friendly fashion in fuel cells. This thesis explores catalysts for oxygen electrocatalysis and how carefully designed local structures on catalysts surfaces termed special active sites can influence the activity. Density functional theory has been used as a method...... is used to explain the increase in activity observed for the OER catalyst ruthenium dioxide when it is mixed with nickel or cobalt. Manganese and cobalt oxides when in the vicinity of gold also display an increase in OER activity which can be explained by locally created special active sites. Density...

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

Science.gov (United States)

Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

2016-02-23

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

International Nuclear Information System (INIS)

Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

1988-01-01

Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells

Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

Energy Technology Data Exchange (ETDEWEB)

Cochran, J.R. Shyr, L.J.

1998-10-05

Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits.

Regulatory Closure Options for the Residue in the Hanford Site Single-Shell Tanks

International Nuclear Information System (INIS)

Cochran, J.R.; Shyr, L.J.

1998-01-01

Liquid, mixed, high-level radioactive waste (HLW) has been stored in 149 single-shell tanks (SSTS) located in tank farms on the U.S. Department of Energy's (DOE's) Hanford Site. The DOE is developing technologies to retrieve as much remaining HLW as technically possible prior to physically closing the tank farms. In support of the Hanford Tanks Initiative, Sandia National Laboratories has addressed the requirements for the regulatory closure of the radioactive component of any SST residue that may remain after physical closure. There is significant uncertainty about the end state of each of the 149 SSTS; that is, the nature and amount of wastes remaining in the SSTS after retrieval is uncertain. As a means of proceeding in the face of these uncertainties, this report links possible end-states with associated closure options. Requirements for disposal of HLW and low-level radioactive waste (LLW) are reviewed in detail. Incidental waste, which is radioactive waste produced incidental to the further processing of HLW, is then discussed. If the low activity waste (LAW) fraction from the further processing of HLW is determined to be incidental waste, then DOE can dispose of that incidental waste onsite without a license from the U.S. Nuclear Regulatory Commissions (NRC). The NRC has proposed three Incidental Waste Criteria for determining if a LAW fraction is incidental waste. One of the three Criteria is that the LAW fraction should not exceed the NRC's Class C limits

Comparison of long-term stability of containment systems for residues and wastes contaminated with naturally occurring radionuclides at an arid site and two humid sites

International Nuclear Information System (INIS)

Winters, M.; Merry-Libby, P.; Hinchman, R.

1985-01-01

The long-term stability of near-surface containment systems designed for the management of radioactive wastes and residues contaminated with naturally occurring radionuclides are compared at the three different sites. The containment designs are: (1) a diked 8.9-m high mound, including a 3.2-m layered cap at a site (humid) near Lewiston, New York, (2) a 6.8-m-high mound, including a similar 3.2-m cap at a site (humid) near Oak Ridge, Tennessee, and (3) 4.8-m deep trenches with 3.0-m backfilled caps at a site (arid) near Hanford, Washington. Geological, hydrological, and biological factors affecting the long-term (1000-year) integrity of the containment systems at each site are examined, including: erosion, flooding, drought, wildfire, slope and cover failure, plant root penetration, burrowing animals, other soil-forming processes, and land-use changes. For the containment designs evaluated, releases of radon-222 at the arid site are predicted to be several orders of magnitude higher than at the two humid sites - upon initial burial and at 1000 years (after severe erosion). Transfer of wastes containing naturally occurring radionuclides from a humid to an arid environment offers little or no advantage relative to long-term stability of the containment system and has a definite disadvantage in terms of gaseous radioactive releases. 26 references, 3 figures, 4 tables

Phytotoxicity of vulpia residues: III. Biological activity of identified allelochemicals from Vulpia myuros.

Science.gov (United States)

An, M; Pratley, J E; Haig, T

2001-02-01

Twenty compounds identified in vulpia (Vulpia myuros) residues as allelochemicals were individually and collectively tested for biological activity. Each exhibited characteristic allelochemical behavior toward the test plant, i.e., inhibition at high concentrations and stimulation or no effect at low concentrations, but individual activities varied. Allelopathins present in large quantities, such as syringic, vanillic, and succinic acids, possessed low activity, while those present in small quantities, such as catechol and hydrocinnamic acid, possessed strong inhibitory activity. The concept of a phytotoxic strength index was developed for quantifying the biological properties of each individual allelopathin in a concise, comprehensive, and meaningful format. The individual contribution of each allelopathin, assessed by comparing the phytotoxic strength index to the overall toxicity of vulpia residues, was variable according to structure and was influenced by its relative proportion in the residue. The majority of compounds possessed low or medium biological activity and contributed most of the vulpia phytotoxicity, while compounds with high biological activity were in the minority and only present at low concentration. Artificial mixtures of these pure allelochemicals also produced phytotoxicity. There were additive/synergistic effects evident in the properties of these mixtures. One such mixture, formulated from allelochemicals found in the same proportions as occur in vulpia extract, produced stronger activity than another formulated from the same set of compounds but in equal proportions. These results suggest that the exploration of the relative composition of a cluster of allelopathins may be more important than simply focusing on the identification of one or two compounds with strong biological activity and that synergism is fundamental to the understanding of allelopathy.

Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

Directory of Open Access Journals (Sweden)

Hana eUhlíková

2016-02-01

Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-isoforms (with only 1-3 lysine residues.To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of beta-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

Residual activity evaluation: a benchmark between ANITA, FISPACT, FLUKA and PHITS codes

Science.gov (United States)

Firpo, Gabriele; Viberti, Carlo Maria; Ferrari, Anna; Frisoni, Manuela

2017-09-01

The activity of residual nuclides dictates the radiation fields in periodic inspections/repairs (maintenance periods) and dismantling operations (decommissioning phase) of accelerator facilities (i.e., medical, industrial, research) and nuclear reactors. Therefore, the correct prediction of the material activation allows for a more accurate planning of the activities, in line with the ALARA (As Low As Reasonably Achievable) principles. The scope of the present work is to show the results of a comparison between residual total specific activity versus a set of cooling time instants (from zero up to 10 years after irradiation) as obtained by two analytical (FISPACT and ANITA) and two Monte Carlo (FLUKA and PHITS) codes, making use of their default nuclear data libraries. A set of 40 irradiating scenarios is considered, i.e. neutron and proton particles of different energies, ranging from zero to many hundreds MeV, impinging on pure elements or materials of standard composition typically used in industrial applications (namely, AISI SS316 and Portland concrete). In some cases, experimental results were also available for a more thorough benchmark.

LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

Directory of Open Access Journals (Sweden)

April eReynolds

2014-06-01

Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

Science.gov (United States)

Mautner, Henry G.; Bartels, Eva

1970-01-01

p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins

Science.gov (United States)

Lin, Chih-Ying

2018-01-01

Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins. PMID:29381770

A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

Science.gov (United States)

Lam, Sonia Y; Yeung, Rachel C Y; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

2011-03-01

Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

Directory of Open Access Journals (Sweden)

Sonia Y Lam

2011-03-01

Full Text Available Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy.Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

Prediction of residual metabolic activity after treatment in NSCLC patients

International Nuclear Information System (INIS)

Rios Velazquez, Emmanuel; Aerts, Hugo J.W.L.; Oberije, Cary; Ruysscher, Dirk De; Lambin, Philippe

2010-01-01

Purpose. Metabolic response assessment is often used as a surrogate of local failure and survival. Early identification of patients with residual metabolic activity is essential as this enables selection of patients who could potentially benefit from additional therapy. We report on the development of a pre-treatment prediction model for metabolic response using patient, tumor and treatment factors. Methods. One hundred and one patients with inoperable NSCLC (stage I-IV), treated with 3D conformal radical (chemo)-radiotherapy were retrospectively included in this study. All patients received a pre and post-radiotherapy fluorodeoxyglucose positron emission tomography-computed tomography FDG-PET-CT scan. The electronic medical record system and the medical patient charts were reviewed to obtain demographic, clinical, tumor and treatment data. Primary outcome measure was examined using a metabolic response assessment on a post-radiotherapy FDG-PET-CT scan. Radiotherapy was delivered in fractions of 1.8 Gy, twice a day, with a median prescribed dose of 60 Gy. Results. Overall survival was worse in patients with residual metabolic active areas compared with the patients with a complete metabolic response (p=0.0001). In univariate analysis, three variables were significantly associated with residual disease: larger primary gross tumor volume (GTVprimary, p=0.002), higher pre-treatment maximum standardized uptake value (SUV max , p=0.0005) in the primary tumor and shorter overall treatment time (OTT, p=0.046). A multivariate model including GTVprimary, SUV max , equivalent radiation dose at 2 Gy corrected for time (EQD2, T) and OTT yielded an area under the curve assessed by the leave-one-out cross validation of 0.71 (95% CI, 0.65-0.76). Conclusion. Our results confirmed the validity of metabolic response assessment as a surrogate of survival. We developed a multivariate model that is able to identify patients at risk of residual disease. These patients may benefit from

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

DEFF Research Database (Denmark)

Daniel, Bastian; Wallner, Silvia; Steiner, Barbara

2016-01-01

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively....... The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been...... be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation...

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

Science.gov (United States)

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation.

Science.gov (United States)

Mueller, Sandra; Kleinau, Gunnar; Jaeschke, Holger; Paschke, Ralf; Krause, Gerd

2008-06-27

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

International Nuclear Information System (INIS)

Zull, Lawrence M.; Yeniscavich, William

2008-01-01

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

Evaluation of residue-residue contact predictions in CASP9

KAUST Repository

Monastyrskyy, Bohdan

2011-01-01

This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures being the precision in recognizing contacts and the difference between the distribution of distances in the subset of predicted contact pairs versus all pairs of residues in the structure. The emphasis is placed on the prediction of long-range contacts (i.e., contacts between residues separated by at least 24 residues along sequence) in target proteins that cannot be easily modeled by homology. Although there is considerable activity in the field, the current analysis reports no discernable progress since CASP8.

Enhancement of methylbenzene adsorption capacity through cetyl trimethyl ammonium bromide-modified activated carbon derived from Astragalus residue

Science.gov (United States)

Feng, Ningchuan; Zhang, Yumei; Fan, Wei; Zhu, Meilin

2018-02-01

Activated carbon was prepared from astragalus residue by KOH and then treated with cetyl trimethyl ammonium bromide (CTAB) and used for the removal of methylbenzene from aqueous solution. The samples were characterized by FTIR, XRD, SEM and Boehm titration. The results showed that CTAB changed the physicochemical properties of activated carbon significantly. The isotherm adsorption studies of methylbenzene onto the astragalus residue activated carbon (ASC) and CTAB-modified astragalus residue activated carbon (ASCCTAB) were examined by using batch techniques and agreed well with the Langmuir model. The maximum adsorption capacity of ASC and ASC-CTAB for methylbenzene determined from the Langmuir model was183.56 mg/g and 235.18 mg/g, respectively. The results indicated that using CTAB as a modifier for ASC modification could markedly enhance the methylbenzene removal from water.

Insights into the functionality of the putative residues involved in enterocin AS-48 maturation.

Science.gov (United States)

Cebrián, Rubén; Maqueda, Mercedes; Neira, José Luis; Valdivia, Eva; Martínez-Bueno, Manuel; Montalbán-López, Manuel

2010-11-01

AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81(His-1Ile)) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

Directory of Open Access Journals (Sweden)

Jin-Suk Han

2005-01-01

Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Science.gov (United States)

Gray, K A; Dutton, P L; Daldal, F

1994-01-25

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Dashboard applications to monitor experiment activities at sites

Energy Technology Data Exchange (ETDEWEB)

Andreeva, Julia; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciaba, Andrea [CERN, European Organization for Nuclear Research (Switzerland); Belforte, Stefano [INFN Trieste (Italy); Boehm, Max [EDS, an HP Company, Plano, TX (United States); Casajus, Adrian [Universitat de Barcelona (Spain); Flix, Josep [PIC, Port d' Informacio CientIfica, Bellaterra (Spain); Tsaregorodtsev, Andrei, E-mail: Elisa.Lanciotti@cern.c, E-mail: Pablo.Saiz@cern.c [CPPM Marseille (France)

2010-04-01

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

Dashboard applications to monitor experiment activities at sites

International Nuclear Information System (INIS)

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

2010-01-01

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

Logging and Agricultural Residue Supply Curves for the Pacific Northwest

Energy Technology Data Exchange (ETDEWEB)

Kerstetter, James D.; Lyons, John Kim

2001-01-01

This report quantified the volume of logging residues at the county level for current timber harvests. The cost of recovering logging residues was determined for skidding, yearding, loading, chipping and transporting the residues. Supply curves were developed for ten candidate conversion sites in the Pacific Northwest Region. Agricultural field residues were also quantified at the county level using five-year average crop yields. Agronomic constraints were applied to arrive at the volumes available for energy use. Collection costs and transportation costs were determined and supply curves generated for thirteen candidate conversion sites.

TmiRUSite and TmiROSite scripts: searching for mRNA fragments with miRNA binding sites with encoded amino acid residues.

Science.gov (United States)

Berillo, Olga; Régnier, Mireille; Ivashchenko, Anatoly

2014-01-01

microRNAs are small RNA molecules that inhibit the translation of target genes. microRNA binding sites are located in the untranslated regions as well as in the coding domains. We describe TmiRUSite and TmiROSite scripts developed using python as tools for the extraction of nucleotide sequences for miRNA binding sites with their encoded amino acid residue sequences. The scripts allow for retrieving a set of additional sequences at left and at right from the binding site. The scripts presents all received data in table formats that are easy to analyse further. The predicted data finds utility in molecular and evolutionary biology studies. They find use in studying miRNA binding sites in animals and plants. TmiRUSite and TmiROSite scripts are available for free from authors upon request and at https: //sites.google.com/site/malaheenee/downloads for download.

Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

International Nuclear Information System (INIS)

Tommaso, Anne di; Hagen, Jussara; Tompkins, Van; Muniz, Viviane; Dudakovic, Amel; Kitzis, Alain; Ladeveze, Veronique; Quelle, Dawn E.

2009-01-01

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala 14 and Thr 31 ) were found to destabilize the protein while two others (Val 24 and Ala 41 ) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val 24 was required for p53-independent growth suppression whereas multiple residues (Val 24 , Thr 31 , Ala 41 and His 60 ) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

Science.gov (United States)

Kshatri, Aravind S; Gonzalez-Hernandez, Alberto J; Giraldez, Teresa

2018-04-01

BK channels are dually regulated by voltage and Ca 2+ , providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca 2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K + (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca 2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca 2+ . Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca 2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca 2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca 2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca 2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca 2+ binding sites regulating the Ca 2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 Elsevier B.V. All rights reserved.

Physical activity and gastric residuals as biomarkers for region-specific NEC lesions in preterm neonates

DEFF Research Database (Denmark)

Cao, Muqing; Andersen, Anders Daniel; Li, Yanqi

2016-01-01

onset of NEC can be predicted by decreased physical activity during the first few days after birth. Methods: Cesarean-delivered preterm pigs were fed parenteral nutrition and increasing amounts of formula for 5 days after birth (n = 120). Their physical activity was quantified by a continuous camera....... Results: Half of the pigs (48%) showed clear NEC-like lesions on day 5, and these individuals had more adverse clinical symptoms from day 3 but decreased physical activity already from day 2 relative to the unaffected pigs (both p ... physical activity on days 2 and 3, and the increased volume of gastric residuals was specifically related to colon lesions (both p physical activity precedes the clinical symptoms of NEC in the small intestine of preterm pigs, and increased gastric residuals predict NEC...

HEU Measurements of Holdup and Recovered Residue in the Deactivation and Decommissioning Activities of the 321-M Reactor Fuel Fabrication Facility at the Savannah River Site

Energy Technology Data Exchange (ETDEWEB)

DEWBERRY, RAYMOND; SALAYMEH, SALEEM R.; CASELLA, VITO R.; MOORE, FRANK S.

2005-03-11

This paper contains a summary of the holdup and material control and accountability (MC&A) assays conducted for the determination of highly enriched uranium (HEU) in the deactivation and decommissioning (D&D) of Building 321-M at the Savannah River Site (SRS). The 321-M facility was the Reactor Fuel Fabrication Facility at SRS and was used to fabricate HEU fuel assemblies, lithium-aluminum target tubes, neptunium assemblies, and miscellaneous components for the SRS production reactors. The facility operated for more than 35 years. During this time thousands of uranium-aluminum-alloy (U-Al) production reactor fuel tubes were produced. After the facility ceased operations in 1995, all of the easily accessible U-Al was removed from the building, and only residual amounts remained. The bulk of this residue was located in the equipment that generated and handled small U-Al particles and in the exhaust systems for this equipment (e.g., Chip compactor, casting furnaces, log saw, lathes A & B, cyclone separator, Freon{trademark} cart, riser crusher, ...etc). The D&D project is likely to represent an important example for D&D activities across SRS and across the Department of Energy weapons complex. The Savannah River National Laboratory was tasked to conduct holdup assays to quantify the amount of HEU on all components removed from the facility prior to placing in solid waste containers. The U-235 holdup in any single component of process equipment must not exceed 50 g in order to meet the container limit. This limit was imposed to meet criticality requirements of the low level solid waste storage vaults. Thus the holdup measurements were used as guidance to determine if further decontamination of equipment was needed to ensure that the quantity of U-235 did not exceed the 50 g limit and to ensure that the waste met the Waste Acceptance Criteria (WAC) of the solid waste storage vaults. Since HEU is an accountable nuclear material, the holdup assays and assays of recovered

Reliability assessment of underground pipelines under the combined effect of active corrosion and residual stress

International Nuclear Information System (INIS)

Amirat, A.; Mohamed-Chateauneuf, A.; Chaoui, K.

2006-01-01

Lifetime management of underground pipelines is mandatory for safe hydrocarbon transmission and distribution systems. Reliability analysis is recognized as a powerful decision-making tool for risk-based design and maintenance. Both the residual stresses generated during the manufacturing process and in-service corrosion reduce the ability to resist internal and external loading. In this study, the residual stress distribution in large diameter pipes has been characterized experimentally in order to be coupled with the corrosion model. During the pipe lifetime, residual stress relaxation occurs due to the loss of pipe thickness as material layers are consumed by corrosion. The reliability-based assessment of residual stress effects is applied to underground pipelines under a roadway, with and without active corrosion. It has been found that the residual stress greatly increases the failure probability, especially in the early stage of the pipe lifetime

A neural measure of behavioral engagement: task-residual low-frequency blood oxygenation level-dependent activity in the precuneus.

Science.gov (United States)

Zhang, Sheng; Li, Chiang-Shan Ray

2010-01-15

Brain imaging has provided a useful tool to examine the neural processes underlying human cognition. A critical question is whether and how task engagement influences the observed regional brain activations. Here we highlighted this issue and derived a neural measure of task engagement from the task-residual low-frequency blood oxygenation level-dependent (BOLD) activity in the precuneus. Using independent component analysis, we identified brain regions in the default circuit - including the precuneus and medial prefrontal cortex (mPFC) - showing greater activation during resting as compared to task residuals in 33 individuals. Time series correlations with the posterior cingulate cortex as the seed region showed that connectivity with the precuneus was significantly stronger during resting as compared to task residuals. We hypothesized that if the task-residual BOLD activity in the precuneus reflects engagement, it should account for a certain amount of variance in task-related regional brain activation. In an additional experiment of 59 individuals performing a stop signal task, we observed that the fractional amplitude of low-frequency fluctuation (fALFF) of the precuneus but not the mPFC accounted for approximately 10% of the variance in prefrontal activation related to attentional monitoring and response inhibition. Taken together, these results suggest that task-residual fALFF in the precuneus may be a potential indicator of task engagement. This measurement may serve as a useful covariate in identifying motivation-independent neural processes that underlie the pathogenesis of a psychiatric or neurological condition.

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

Science.gov (United States)

Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

2015-01-01

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. PMID:25765648

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

Science.gov (United States)

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Site Transition Process Upon Cleanup Completion. Fact Sheet

International Nuclear Information System (INIS)

2009-01-01

After environmental remediation is completed at a site and there is no continuing mission, responsibility for the site and the associated records are transferred to the U.S. Department of Energy (DOE) Office of Legacy Management for post-closure management. Where residual hazards (e.g., disposal cells, ground water contamination) remain, active long-term surveillance and maintenance will be required to ensure protection of human health and the environment

A highly Conserved Aspartic Acid Residue of the Chitosanase from Bacillus Sp. TS Is Involved in the Substrate Binding.

Science.gov (United States)

Zhou, Zhanping; Zhao, Shuangzhi; Liu, Yang; Chang, Zhengying; Ma, Yanhe; Li, Jian; Song, Jiangning

2016-11-01

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

Disposal of residue from uranium ore processing in France

International Nuclear Information System (INIS)

Crochon, Ph.

2011-01-01

Between 1949 and 2001, French mines produced 76, 000 metric tons of uranium and 50 million metric tons of ore, processing residues are stored at 17 sites (in ponds enclosed by dykes or in former open-cast mines) subject to ICPE (classified facility for environment protection) regulation. These disposal sites cover surface areas of between one and several tens of hectares and several thousands to several millions of metric tons of waste are stored at them. When uranium mining stopped in France, these sites were redeveloped, with caps placed over the residue to provide mechanical and radiological protection. All these sites are still monitored by AREVA. In the last fifteen years, these sites have been the subject of a number of studies, especially regarding the long-term evolution and impact of the residue. These studies are now being pursued within the framework of the national plan for the management of nuclear materials and waste (PNGMDR). A regulatory and institutional framework regarding long-term management of these disposal sites needs to be defined. (author)

Characterization of Solids in Residual Wastes from Single-Shell Tanks at the Hanford Site, Washington, USA - 9277

International Nuclear Information System (INIS)

Krupka, Kenneth M.; Cantrell, Kirk J.; Schaef, Herbert T.; Arey, Bruce W.; Heald, Steve M.; Deutsch, William J.; Lindberg, Michael J.

2009-01-01

Solid-phase characterization methods have been used in an ongoing study of residual wastes (i.e., waste remaining after final retrieval operations) from the underground single-shell storage tanks 241-C-103, 241-C-106, 241-C-202, 241-C-203, and 241-S-112 at the U.S. Department of Energy's Hanford Site in Washington State. The results of studies completed to date show significant variability in the compositions of those residual wastes and the compositions, morphologies, and crystallinities of the individual phases that make up these wastes. These differences undoubtedly result from the various waste types stored and transferred in and out each tank and the sluicing and retrieval operations used for waste retrieval. Our studies indicate that these residual wastes are chemically-complex assemblages of crystalline and amorphous solids that contain contaminants as discrete phases and/or co-precipitated within oxide phases. Depending on the specific tank, various solids (e.g., gibbsite; boehmite; dawsonite; cancrinite; Fe oxides such as hematite, goethite, and maghemite; rhodochrosite; lindbergite; whewellite; nitratine; and numerous amorphous or poorly crystalline phases) have been identified by X-ray diffraction and scanning electron microscopy/energy dispersive X-ray spectroscopy in residual wastes studied to date. Our studies also show that contact of residual wastes with Ca(OH)2- and CaCO3-saturated aqueous solutions, which were used as surrogates for the compositions of pore-fluid leachants derived from young and aged cements respectively, may alter the compositions of solid phases present in the contacted wastes. Fe oxides/hydroxides have been identified in all residual wastes studied to date. They occur in these wastes as discrete particles, particles intergrown within a matrix of other phases, and surface coatings on other particles or particle aggregates. These Fe oxides/hydroxides typically contain trace concentrations of other transition metals, such Cr, Mn

Safety aspects of targets for ADTT: Activity, volatile products, residual heat release

International Nuclear Information System (INIS)

Gai, E.V.; Ignatyuk, A.V.; Lunev, V.P.; Shubin, Yu.N.

1999-01-01

Safety aspects of heavy metal liquid targets for the accelerator driven systems connected with the activity accumulation and residual energy release due to the irradiation with high energy proton beam are discussed. The results obtained for the lead-bismuth target that are under construction in IPPE now in the frame of ISTC Project No. 559 are briefly presented. The calculations and the analysis of the accumulation of the spallation reaction products, activity and energy release at various moments after the accelerator shutdown are presented. The concentrations of the reaction products, the total and partial activities, the activities of volatile products are determined. The contributions of the short-lived nuclides important for the prediction of the facility behaviour in regimes with the accelerator beam trips. The calculations and analysis of the residual energy release due to different decay type have been performed. The conclusions are as follows. The obtained results showed that long lived radioactivity accumulates mainly due to primary nuclear reactions. Secondary reactions are responsible for the production of small number of long-lived isotopes Bi-207, Po-210 and some others, being generated by radiative capture of low energy neutrons. It is possible to make a conclusion that neutrons in the energy range 20 - 800 MeV and protons with energy above 100 MeV give main contribution to the total activity generation although these parts of spectra inside the target give comparatively small contribution to the total flux. The correct consideration of short-lived nuclides contribution is the main problem in the analysis of the target behaviour in the case of short accelerator shutdowns. They make the determining contribution to the both activity and the heat release at the first moments after the accelerator shutdown, creating the intermediate links and additional channels for the long-lived nuclides accumulation chains. The strong dependence of calculated

Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs.

Science.gov (United States)

Gomez, Ana; Cardoso, Christiane; Genta, Fernando A; Terra, Walter R; Ferreira, Clélia

2013-08-01

The soluble midgut trehalase from Tenebrio molitor (TmTre1) was purified after several chromatographic steps, resulting in an enzyme with 58 kDa and pH optimum 5.3 (ionizing active groups in the free enzyme: pK(e1) = 3.8 ± 0.2 pK(e2) = 7.4 ± 0.2). The purified enzyme corresponds to the deduced amino acid sequence of a cloned cDNA (TmTre1-cDNA), because a single cDNA coding a soluble trehalase was found in the T. molitor midgut transcriptome. Furthermore, the mass of the protein predicted to be coded by TmTre1-cDNA agrees with that of the purified enzyme. TmTre1 has the essential catalytic groups Asp 315 and Glu 513 and the essential Arg residues R164, R217, R282. Carbodiimide inactivation of the purified enzyme at different pH values reveals an essential carboxyl group with pKa = 3.5 ± 0.3. Phenylglyoxal modified a single Arg residue with pKa = 7.5 ± 0.2, as observed in the soluble trehalase from Spodoptera frugiperda (SfTre1). Diethylpyrocarbonate modified a His residue that resulted in a less active enzyme with pK(e1) changed to 4.8 ± 0.2. In TmTre1 the modified His residue (putatively His 336) is more exposed than the His modified in SfTre1 (putatively His 210) and that affects the ionization of an Arg residue. The architecture of the active site of TmTre1 and SfTre1 is different, as shown by multiple inhibition analysis, the meaning of which demands further research. Trehalase sequences obtained from midgut transcriptomes (pyrosequencing and Illumina data) from 8 insects pertaining to 5 different orders were used in a cladogram, together with other representative sequences. The data suggest that the trehalase gene went duplication and divergence prior to the separation of the paraneopteran and holometabolan orders and that the soluble trehalase derived from the membrane-bound one by losing the C-terminal transmembrane loop. Copyright © 2013 Elsevier Ltd. All rights reserved.

Novel acetylcholinesterase target site for malaria mosquito control.

Directory of Open Access Journals (Sweden)

Yuan-Ping Pang

2006-12-01

Full Text Available Current anticholinesterase pesticides were developed during World War II and are toxic to mammals because they target a catalytic serine residue of acetylcholinesterases (AChEs in insects and in mammals. A sequence analysis of AChEs from 73 species and a three-dimensional model of a malaria-carrying mosquito (Anopheles gambiae AChE (AgAChE reported here show that C286 and R339 of AgAChE are conserved at the opening of the active site of AChEs in 17 invertebrate and four insect species, respectively. Both residues are absent in the active site of AChEs of human, monkey, dog, cat, cattle, rabbit, rat, and mouse. The 17 invertebrates include house mosquito, Japanese encephalitis mosquito, African malaria mosquito, German cockroach, Florida lancelet, rice leaf beetle, African bollworm, beet armyworm, codling moth, diamondback moth, domestic silkworm, honey bee, oat or wheat aphid, the greenbug, melon or cotton aphid, green peach aphid, and English grain aphid. The four insects are house mosquito, Japanese encephalitis mosquito, African malaria mosquito, and German cockroach. The discovery of the two invertebrate-specific residues enables the development of effective and safer pesticides that target the residues present only in mosquito AChEs rather than the ubiquitous serine residue, thus potentially offering an effective control of mosquito-borne malaria. Anti-AgAChE pesticides can be designed to interact with R339 and subsequently covalently bond to C286. Such pesticides would be toxic to mosquitoes but not to mammals.

LIBRA: LIgand Binding site Recognition Application.

Science.gov (United States)

Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

2015-12-15

In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Computational learning on specificity-determining residue-nucleotide interactions

KAUST Repository

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Moses, Alan M.; Zhang, Zhaolei

2015-01-01

The protein–DNA interactions between transcription factors and transcription factor binding sites are essential activities in gene regulation. To decipher the binding codes, it is a long-standing challenge to understand the binding mechanism across different transcription factor DNA binding families. Past computational learning studies usually focus on learning and predicting the DNA binding residues on protein side. Taking into account both sides (protein and DNA), we propose and describe a computational study for learning the specificity-determining residue-nucleotide interactions of different known DNA-binding domain families. The proposed learning models are compared to state-of-the-art models comprehensively, demonstrating its competitive learning performance. In addition, we describe and propose two applications which demonstrate how the learnt models can provide meaningful insights into protein–DNA interactions across different DNA binding families.

Computational learning on specificity-determining residue-nucleotide interactions

KAUST Repository

Wong, Ka-Chun

2015-11-02

The protein–DNA interactions between transcription factors and transcription factor binding sites are essential activities in gene regulation. To decipher the binding codes, it is a long-standing challenge to understand the binding mechanism across different transcription factor DNA binding families. Past computational learning studies usually focus on learning and predicting the DNA binding residues on protein side. Taking into account both sides (protein and DNA), we propose and describe a computational study for learning the specificity-determining residue-nucleotide interactions of different known DNA-binding domain families. The proposed learning models are compared to state-of-the-art models comprehensively, demonstrating its competitive learning performance. In addition, we describe and propose two applications which demonstrate how the learnt models can provide meaningful insights into protein–DNA interactions across different DNA binding families.

Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

Energy Technology Data Exchange (ETDEWEB)

Fardini, Yann [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France); Perez-Cervera, Yobana [Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d' Ascq (France); Facultad de Odontología, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca (Mexico); Camoin, Luc [INSERM, U1068, CRCM, Marseille Protéomique IBiSA, Marseille, F-13009 (France); Institut Paoli-Calmettes Team, Cell Polarity, Cell Signaling and Cancer, Marseille, F-13009 (France); Aix-Marseille Université, F-13284, Marseille (France); CNRS, UMR7258, CRCM, Marseille, F-13009 (France); Pagesy, Patrick [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France); Lefebvre, Tony [Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d' Ascq (France); Issad, Tarik, E-mail: tarik.issad@inserm.fr [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France)

2015-06-26

O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified. - Highlights: • We mutate four previously identified O-GlcNAcylation sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity.

Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

International Nuclear Information System (INIS)

Fardini, Yann; Perez-Cervera, Yobana; Camoin, Luc; Pagesy, Patrick; Lefebvre, Tony; Issad, Tarik

2015-01-01

O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified. - Highlights: • We mutate four previously identified O-GlcNAcylation sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-04-02

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

An overview of the prediction of protein DNA-binding sites.

Science.gov (United States)

Si, Jingna; Zhao, Rui; Wu, Rongling

2015-03-06

Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

Environmental dredging residual generation and management.

Science.gov (United States)

Patmont, Clay; LaRosa, Paul; Narayanan, Raghav; Forrest, Casey

2018-05-01

The presence and magnitude of sediment contamination remaining in a completed dredge area can often dictate the success of an environmental dredging project. The need to better understand and manage this remaining contamination, referred to as "postdredging residuals," has increasingly been recognized by practitioners and investigators. Based on recent dredging projects with robust characterization programs, it is now understood that the residual contamination layer in the postdredging sediment comprises a mixture of contaminated sediments that originate from throughout the dredge cut. This mixture of contaminated sediments initially exhibits fluid mud properties that can contribute to sediment transport and contamination risk outside of the dredge area. This article reviews robust dredging residual evaluations recently performed in the United States and Canada, including the Hudson River, Lower Fox River, Ashtabula River, and Esquimalt Harbour, along with other projects. These data better inform the understanding of residuals generation, leading to improved models of dredging residual formation to inform remedy evaluation, selection, design, and implementation. Data from these projects confirm that the magnitude of dredging residuals is largely determined by site conditions, primarily in situ sediment fluidity or liquidity as measured by dry bulk density. While the generation of dredging residuals cannot be avoided, residuals can be successfully and efficiently managed through careful development and implementation of site-specific management plans. Integr Environ Assess Manag 2018;14:335-343. © 2018 The Authors. Integrated Environmental Assessment and Management Published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC). © 2018 The Authors. Integrated Environmental Assessment and Management Published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

[The importance of C-terminal aspartic acid residue (D141) to the antirestriction activity of the ArdB (R64) protein].

Science.gov (United States)

Kudryavtseva, A A; Osetrova, M S; Livinyuk, V Ya; Manukhov, I V; Zavilgelsky, G B

2017-01-01

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.

Evaluation and optimization of nutritional and environmental impact of biogas residues

International Nuclear Information System (INIS)

Lichti, Fabian Heribert

2013-01-01

On the basis of the dynamic growth of biogas plants in Germany the fertilization with biogas residues has obtained an important role for recirculation of plant nutrients, particularly with regard to nitrogen. In this work the effect of N nutrition with biogas residues was assessed in a 3-year on-field trial conducted at four sites throughout Bavaria. The fertilizing effects were tested by varying rate and time of biogas residues application, using different application techniques and the addition of nitrification inhibitors on several crops. The biogas residues achieved mineral fertilizer equivalents of 30 - 45 %. Overall, the untreated biogas residues showed a slightly increased N efficiency compared to cattle manure, whereas particularly site-dependent differences resulted in large differences in N efficiency of biogas residues.

Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Pegan, Scott D. [Univ. of Denver, CO (United States); Rukseree, Kamolchanok [National Center for Genetic Engineering and Biotechnology (BIOTEC), Tha Khlong (Thailand); Capodagli, Glenn C. [Univ. of Denver, CO (United States); Baker, Erica A. [Univ. of Denver, CO (United States); Krasnykh, Olga [Univ. of Illinois, Chicago, IL (United States); Franzblau, Scott G. [Univ. of Illinois, Chicago, IL (United States); Mesecar, Andrew D. [Purdue Univ., West Lafayette, IN (United States)

2013-01-08

The class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprises one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation–deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA–PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation–protonation step of the MtFBA reaction mechanism. Furthermore, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

Science.gov (United States)

Sánchez Centellas, Daniel; Gudlur, Sushanth; Vicente-Carrillo, Alejandro; Ramström, Sofia; Lindahl, Tomas L

2017-06-01

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased β-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantum mechanical design of enzyme active sites.

Science.gov (United States)

Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

2008-02-01

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox.

Directory of Open Access Journals (Sweden)

Julien Hiblot

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

Self-activation of biochar from furfural residues by recycled pyrolysis gas.

Science.gov (United States)

Yin, Yulei; Gao, Yuan; Li, Aimin

2018-04-17

Biochar samples with controllable specific surface area and mesopore ratio were self-activated from furfural residues by recycled pyrolysis gas. The objective of this study was to develop a new cyclic utilization method for the gas produced by pyrolysis. The influences of preparation parameters on the resulting biochar were studied by varying the pyrolysis-gas flow rate, activation time and temperature. Structural characterization of the produced biochar was performed by analysis of nitrogen adsorption isotherms at 77 K and scanning electron microscope (SEM). The pyrolysis gas compositions before and after activation were determined by a gas chromatograph. The results indicated that the surface area of the biochar was increased from 167 m 2 /g to 567 m 2 /g, the total pore volume increased from 0.121 cm 3 /g to 0.380 cm 3 /g, and the ratio of the mesopore pore volume to the total pore volume increased 17-39.7%. The CO volume fraction of the pyrolysis gas changed from 34.66 to 62.29% and the CO 2 volume fraction decreased from 48.26% to 12.17% under different conditions of pyrolysis-gas flow rate, activation time and temperature. The calorific values of pyrolysis gas changed from 8.82 J/cm 3 to 14.00 J/cm 3 , which were higher than those of conventional pyrolysis gases. The slower pyrolysis-gas flow rate and higher activation time increased the efficiency of the reaction between carbon and pyrolysis gas. These results demonstrated the feasibility of treatment of the furfural residues to produce microporous and mesoporous biochar. The pyrolysis gas that results from the activation process could be used as fuel. Overall, this new self-activation method meets the development requirements of cyclic economy and cleaner production. Copyright © 2018. Published by Elsevier Ltd.

A method for the determination of residual beta activity in drinking water samples

Energy Technology Data Exchange (ETDEWEB)

Idoeta, R. [Dpto. Ingenieria Nuclear y Mecanica de Fluidos, E. T. S. Ingenieria de Bilbao - Universidad del Pais Vasco (UPV/EHU), Alda. Urquijo s/n. 48013 Bilbao (Spain)], E-mail: raquel.idoeta@ehu.es; Herranz, M.; Abelairas, A.; Legarda, F. [Dpto. Ingenieria Nuclear y Mecanica de Fluidos, E. T. S. Ingenieria de Bilbao - Universidad del Pais Vasco (UPV/EHU), Alda. Urquijo s/n. 48013 Bilbao (Spain)

2007-09-15

The determination of residual beta activity in drinking water is usually needed in most monitoring programs. In this work a procedure for its determination is described and expressions for the calculations of detection limits and uncertainties are proposed.

A method for the determination of residual beta activity in drinking water samples

International Nuclear Information System (INIS)

Idoeta, R.; Herranz, M.; Abelairas, A.; Legarda, F.

2007-01-01

The determination of residual beta activity in drinking water is usually needed in most monitoring programs. In this work a procedure for its determination is described and expressions for the calculations of detection limits and uncertainties are proposed

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

Science.gov (United States)

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

Probing the putative active site of YjdL

DEFF Research Database (Denmark)

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

2012-01-01

pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278...... with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub...

Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1.

Science.gov (United States)

Hamid, Azzmer Azzar Abdul; Hamid, Tengku Haziyamin Tengku Abdul; Wahab, Roswanira Abdul; Huyop, Fahrul

2015-03-01

The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distribution patterns of firearm discharge residues as revealed by neutron activation analysis

International Nuclear Information System (INIS)

Pillay, K.K.S.; Driscoll, D.C.; Jester, W.A.

1975-01-01

A systematic investigation using a variety of handguns has revealed the existence of distinguisable distribution patterns of firearm discharge residues on surfaces below the flight path of a bullet. The residues are identificable even at distances of 12 meters from the gun using nondestructive neutron activation analysis. The results of these investigations show that the distribution pattern for a gun is reproducible using similar ammunition and that there exist two distinct regions to the patterns developed between the firearm and the target-one with respect to the position of the gun and the other in the vicinity of the target. The judicious applications of these findings could be of significant value in criminal investigations. (T.G.)

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

DEFF Research Database (Denmark)

Chen, Z. W.; Jiang, C. Y.; She, Qunxin

2005-01-01

). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant......Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system...... proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have...

Probing the emitter site of Renilla luciferase using small organic molecules; an attempt to understand the molecular architecture of the emitter site.

Science.gov (United States)

Salehi, Farajollah; Emamzadeh, Rahman; Nazari, Mahboobeh; Rasa, Seyed Mohammad Mahdi

2016-12-01

Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6-66mM) and isopropanol (19.3-76mM) the K m changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the "non-binding" model was proposed. Copyright © 2016 Elsevier B.V. All rights reserved.

Remaining Sites Verification Package for the 600-233 Waste Site, Vertical Pipe Near 100-B Electrical Laydown Area. Attachment to Waste Site Reclassification Form 2005-041

International Nuclear Information System (INIS)

Carlson, R.A.

2005-01-01

The 600-233 waste site consisted of three small-diameter pipelines within the 600-232 waste site, including previously unknown diesel fuel supply lines discovered during site remediation. The 600-233 waste site has been remediated to achieve the remedial action objectives specified in the Remaining Sites ROD. The results of verification sampling show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River

Characterization of the heterotrophic biomass and the endogenous residue of activated sludge.

Science.gov (United States)

Ramdani, Abdellah; Dold, Peter; Gadbois, Alain; Déléris, Stéphane; Houweling, Dwight; Comeau, Yves

2012-03-01

The activated sludge process generates an endogenous residue (X(E)) as a result of heterotrophic biomass decay (X(H)). A literature review yielded limited information on the differences between X(E) and X(H) in terms of chemical composition and content of extracellular polymeric substances (EPS). The objective of this project was to characterize the chemical composition (x, y, z, a, b and c in C(x)H(y)O(z)N(a)P(b)S(c)) of the endogenous and the active fractions and EPS of activated sludge from well designed experiments. To isolate X(H) and X(E) in this study, activated sludge was generated in a 200L pilot-scale aerobic membrane bioreactor (MBR) fed with a soluble and completely biodegradable synthetic influent of sodium acetate as the sole carbon source. This influent, which contained no influent unbiodegradable organic or inorganic particulate matter, allowed the generation of a sludge composed essentially of two fractions: heterotrophic biomass X(H) and an endogenous residue X(E), the nitrifying biomass being negligible. The endogenous decay rate and the active biomass fraction of the MBR sludge were determined in 21-day aerobic digestion batch tests by monitoring the VSS and OUR responses. Fractions of X(H) and X(E) were respectively 68% and 32% in run 1 (MBR at 5.2 day SRT) and 59% and 41% in run 2 (MBR at 10.4 day SRT). The endogenous residue was isolated by subjecting the MBR sludge to prolonged aerobic batch digestion for 3 weeks, and was characterized in terms of (a) elemental analysis for carbon, nitrogen, phosphorus and sulphur; and (b) content of EPS. The MBR sludge was characterized using the same procedures (a and b). Knowing the proportions of X(H) and X(E) in this sludge, it was possible to characterize X(H) by back calculation. Results from this investigation showed that the endogenous residue had a chemical composition different from that of the active biomass with a lower content of inorganic matter (1:4.2), of nitrogen (1:2.9), of phosphorus (1

Effect of the replacement of aspartic acid/glutamic acid residues with asparagine/glutamine residues in RNase He1 from Hericium erinaceus on inhibition of human leukemia cell line proliferation.

Science.gov (United States)

Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Suzuki, Mamoru; Inokuchi, Norio

2015-01-01

RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity.

Federal Facility Compliance Act, Proposed Site Treatment Plan: Background Volume. Executive Summary

International Nuclear Information System (INIS)

1995-01-01

This Federal Facility Compliance Act Site Treatment Plan discusses the options of radioactive waste management for Ames Laboratory. This is the background volume which discusses: site history and mission; framework for developing site treatment plans; proposed plan organization and related activities; characterization of mixed waste and waste minimization; low level mixed waste streams and the proposed treatment approach; future generation of TRU and mixed wastes; the adequacy of mixed waste storage facilities; and a summary of the overall DOE activity in the area of disposal of mixed waste treatment residuals

Active site of mycobacterial dUTPase: Structural characteristics and a built-in sensor

International Nuclear Information System (INIS)

Varga, Balazs; Barabas, Orsolya; Takacs, Eniko; Nagy, Nikolett; Nagy, Peter; Vertessy, Beata G.

2008-01-01

dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, α,β-imido-dUTP and Mg 2+ at 1.5 A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K d for α,β-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed

Review of the incineration of 500 tonnes of radio-active residues

International Nuclear Information System (INIS)

Rodier, J.; Seyfried, P.; Charbonneaux, M.

1969-01-01

During its first five years operation, the incinerator at the Marcoule Centre has burnt almost 500 tonnes of radio-active residues. Improvements in some of the details of the process have been made during this period; they concern the nature of the materials involved. The technical and radiological results for the installation are very favorable, and have made it possible to maintain a high charge factor.Although the overall economic results are not advantageous in the case of ungraded solid residues this method represents nevertheless the best available for eliminating oils, solvents, wood and dead animals. It can also be of use furthermore each time that a dilution in the atmosphere can advantageously be used as a method of disposing of certain radio elements such as tritium or carbon 14 in the form of gases or vapours. (author) [fr

The active site of hen egg-white lysozyme: flexibility and chemical bonding

Energy Technology Data Exchange (ETDEWEB)

Held, Jeanette, E-mail: jeanette.netzel@uni-bayreuth.de; Smaalen, Sander van [University of Bayreuth, D-95440 Bayreuth (Germany)

2014-04-01

Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters. Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C�

The active site of hen egg-white lysozyme: flexibility and chemical bonding

International Nuclear Information System (INIS)

Held, Jeanette; Smaalen, Sander van

2014-01-01

Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters. Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C�

Text mining improves prediction of protein functional sites.

Directory of Open Access Journals (Sweden)

Karin M Verspoor

Full Text Available 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.

Text Mining Improves Prediction of Protein Functional Sites

Science.gov (United States)

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

Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor

Directory of Open Access Journals (Sweden)

Igor E. Deyev

2017-11-01

Full Text Available The orphan insulin receptor-related receptor (IRR, in contrast to its close homologs, the insulin receptor (IR and insulin-like growth factor receptor (IGF-IR can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646–716 within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

The selenazal drug ebselen potently inhibits indoleamine 2,3-dioxygenase by targeting enzyme cysteine residues.

Science.gov (United States)

Terentis, Andrew C; Freewan, Mohammed; Sempértegui Plaza, Tito S; Raftery, Mark J; Stocker, Roland; Thomas, Shane R

2010-01-26

The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory role by catalyzing the oxidative degradation of l-tryptophan. Here we show that the selenezal drug ebselen is a potent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a manner independent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDO (rIDO) with an apparent inhibition constant of 94 +/- 17 nM. Optical and resonance Raman spectroscopy showed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron from the predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch of the CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed that ebselen enhanced nonproductive l-tryptophan binding, while circular dichroism indicated that the drug reduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed that ebselen reacted with multiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitol reversed the effects of the drug on the heme environment and significantly restored enzyme activity. These findings indicate that ebselen inhibits IDO activity by reacting with the enzyme's cysteine residues that result in changes to protein conformation and active site heme, leading to an increase in the level of nonproductive substrate binding. This study highlights that modification of cysteine residues is a novel and effective means of inhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents a previously unrecognized in vivo target of ebselen.

Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

Science.gov (United States)

Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

2018-04-16

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

Science.gov (United States)

Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

1995-02-27

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

Residual limb skin temperature and thermal comfort in people with amputation during activity in a cold environment.

Science.gov (United States)

Segal, Ava D; Klute, Glenn K

2016-01-01

Thermal comfort remains a common problem for people with lower-limb amputation. Both donning a prosthesis and engaging in activity at room temperature can increase residual limb skin temperature; however, the effects of activity on skin temperature and comfort in more extreme environments remain unknown. We examined residual limb skin temperatures and perceived thermal comfort (PTC; 11-point Likert scale) of participants with unilateral transtibial amputation (n = 8) who were snowshoeing in a cold environment. Residual limb skin temperature increased by 3.9°C [3.0°C to 4.7°C] (mean difference [95% confidence interval (CI)], p < 0.001) after two 30 min exercise sessions separated by a 5 min rest session. Minimal cooling (-0.2°C [-1.1°C to 0.6°C]) occurred during the rest period. Similar changes in PTC were found for the residual limb, intact limb, and whole body, with a mean scale increase of 1.6 [1.1 to 2.1] and 1.3 [0.8 to 1.8] for the first and second exercise sessions, respectively (p < 0.001). Activity in a cold environment caused similar increases in residual limb skin temperature as those found in studies conducted at room temperature. Participants with amputation perceived warming as their skin temperature increased during exercise followed by the perception of cooling during rest, despite minimal associated decreases in skin temperature.

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

Science.gov (United States)

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

Science.gov (United States)

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Remaining Sites Verification Package for 100-F-38 Stained Soil Site. Attachment to Waste Site Reclassification Form 2004-093

International Nuclear Information System (INIS)

Carlson, R.A.

2006-01-01

The 100-F-38 Stained Soil site was an area of yellow stained soil that was discovered while excavating a trench for the placement of electrical conduit. The 100-F-38 Stained Soil site meets the remedial action objectives specified in the Remaining Sites ROD. The results of verification sampling show demonstrate that residual contaminant concentrations support future unrestricted land uses that can be represented by a rural-residential scenario. The results also show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils and the contaminant concentrations remaining in the soil are protective of groundwater and the Columbia River

Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases.

Science.gov (United States)

Buchner, John M; Robertson, Anne E; Poynter, David J; Denniston, Shelby S; Karls, Anna C

2005-05-01

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

Salt site performance assessment activities

International Nuclear Information System (INIS)

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

Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity.

Directory of Open Access Journals (Sweden)

Shotaro Sasaki

Full Text Available Monocarboxylate transporter 4 (MCT4 is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity.

Adsorption and oxidation of SO₂in a fixed-bed reactor using activated carbon produced from oxytetracycline bacterial residue and impregnated with copper.

Science.gov (United States)

Zhou, Baohua; Yu, Lei; Song, Hanning; Li, Yaqi; Zhang, Peng; Guo, Bin; Duan, Erhong

2015-02-01

The SO₂removal ability (including adsorption and oxidation ability) of activated carbon produced from oxytetracycline bacterial residue and impregnated with copper was investigated. The activated carbon produced from oxytetracycline bacterial residue and modified with copper was characterized by x-ray diffraction, scanning electron microscopy, and energy-dispersive spectroscopy. The effects of the catalysts, SO₂concentration, weight hourly space velocity, and temperature on the SO₂adsorption and oxidation activity were evaluated. Activated carbon produced from oxytetracycline bacterial residue and used as catalyst supports for copper oxide catalysts provided high catalytic activity for the adsorbing and oxidizing of SO₂from flue gases.

Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations

Directory of Open Access Journals (Sweden)

Longcan Mei

2018-03-01

Full Text Available A superkine variant of interleukin-2 with six site mutations away from the binding interface developed from the yeast display technique has been previously characterized as undergoing a distal structure alteration which is responsible for its super-potency and provides an elegant case study with which to get insight about how to utilize allosteric effect to achieve desirable protein functions. By examining the dynamic network and the allosteric pathways related to those mutated residues using various computational approaches, we found that nanosecond time scale all-atom molecular dynamics simulations can identify the dynamic network as efficient as an ensemble algorithm. The differentiated pathways for the six core residues form a dynamic network that outlines the area of structure alteration. The results offer potentials of using affordable computing power to predict allosteric structure of mutants in knowledge-based mutagenesis.

Active disease and residual damage in treated Wegener's granulomatosis: an observational study using pulmonary high-resolution computed tomography

International Nuclear Information System (INIS)

Komocsi, Andras; Reuter, Michael; Heller, Martin; Murakoezi, Henriette; Gross, Wolfgang L.; Schnabel, Armin

2003-01-01

The purpose of this study was to determine to what extent high-resolution computed tomography (HRCT) of the lungs can distinguish active inflammatory disease from inactive cicatricial disease in patients treated for Wegener's granulomatosis (WG). Twenty-eight WG patients with active pulmonary disease underwent a first HRCT examination immediately before standard immunosuppressive treatment and a second examination after clinical remission had been achieved. Lesions remaining after treatment were categorized as residual damage and were compared with findings during active disease to see by what features active and cicatricial disease can be distinguished. During active disease 17 patients had nodules/masses, 12 had ground-glass opacities, 6 had septal lines and 6 had non-septal lines. After treatment, ground-glass opacities had resolved completely. Nodules/masses had resolved in 8 patients and had diminished in 7 patients. Residual nodules were distinguished from nodules/masses in active disease by lack of cavitation and a diameter of mostly <15 mm. In one-third of patients lines resolved, but in 8 instances new lines evolved during immunosuppression. During a follow-up period of a median 26.5 months (range 20.0-33.8), patients with residual nodules or lines had no more relapses than patients with completely cleared lungs. Treated pulmonary WG leaves substantial residual damage. High-resolution CT does assist in the distinction between active and inactive lesions. Ground-glass opacities, cavitating nodules/masses and masses measuring more than 3 cm represent active disease ordinarily. Non-cavitary small nodules and septal or non-septal lines can be either active or cicatricial lesions. The nature of these lesions needs to be clarified by longitudinal observation. (orig.)

Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

Science.gov (United States)

Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

2014-07-23

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

Using residual stacking to mitigate site-specific errors in order to improve the quality of GNSS-based coordinate time series of CORS

Science.gov (United States)

Knöpfler, Andreas; Mayer, Michael; Heck, Bernhard

2014-05-01

Within the last decades, positioning using GNSS (Global Navigation Satellite Systems; e.g., GPS) has become a standard tool in many (geo-) sciences. The positioning methods Precise Point Positioning and differential point positioning based on carrier phase observations have been developed for a broad variety of applications with different demands for example on accuracy. In high precision applications, a lot of effort was invested to mitigate different error sources: the products for satellite orbits and satellite clocks were improved; the misbehaviour of satellite and receiver antennas compared to an ideal antenna is modelled by calibration values on absolute level, the modelling of the ionosphere and the troposphere is updated year by year. Therefore, within processing of data of CORS (continuously operating reference sites), equipped with geodetic hardware using a sophisticated strategy, the latest products and models nowadays enable positioning accuracies at low mm level. Despite the considerable improvements that have been achieved within GNSS data processing, a generally valid multipath model is still lacking. Therefore, site specific multipath still represents a major error source in precise GNSS positioning. Furthermore, the calibration information of receiving GNSS antennas, which is for instance derived by a robot or chamber calibration, is valid strictly speaking only for the location of the calibration. The calibrated antenna can show a slightly different behaviour at the CORS due to near field multipath effects. One very promising strategy to mitigate multipath effects as well as imperfectly calibrated receiver antennas is to stack observation residuals of several days, thereby, multipath-loaded observation residuals are analysed for example with respect to signal direction, to find and reduce systematic constituents. This presentation will give a short overview about existing stacking approaches. In addition, first results of the stacking approach

[Adenylate cyclase from rabbit heart: substrate binding site].

Science.gov (United States)

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

Remaining Sites Verification Package for the 100-D-2 Lead Sheeting Waste Site. Attachment to Waste Site Reclassification Form 2007-030

International Nuclear Information System (INIS)

Dittmer, L.M.

2008-01-01

The 100-D-2 Lead Sheeting waste site was located approximately 50 m southwest of the 185-D Building and approximately 16 m north of the east/west oriented road. The site consisted of a lead sheet covering a concrete pad. In accordance with this evaluation, the verification sampling results support a reclassification of this site to Interim Closed Out. The results of verification sampling show that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River

Decomposition of dilute residual active chlorine in sea-water

International Nuclear Information System (INIS)

Yoshinaga, Tetsutaro; Kawano, Kentaro; Yanagase, Kenjiro; Shiga, Akira

1985-01-01

Coastal industries such as power stations require enormous quantities of sea-water for cooling, but the marine organisms in it often result in fouling and/or blockade of the circulating water condenser and pipeworks. To prevent this, chlorine, or hypochlorite by the direct electrolysis of sea-water have been added. Environmental concerns, however, dictate that the residual chlorine concentration at the outlet should be less than the regulated value (0.02 ppm). Methods for decomposing dilute residual chlorine solutions were therefore studied. It was found that: 1) The addition of (raw) sea-water to the sea-water which passed through the condenser lowered the residual chlorine concentration to an greater extent than could be expected by dilution only. 2) Ozonation of the residual chlorine solution led to degradation of OCl - , but in solutions with a residual chlorine concentrations of less than 3 -- 4 ppm, ozonation had no effect. 3) Irradiation with ultra violet light (254 nm) decomposed the residual chlorine. Under the present work conditions (25 0 C: pH 8; depth 10 mm), nearly first order kinetics were to hold [da/dt = ksub((1)) (1-a)sup(n)]. There is a proportional relationship between the kinetic constant (k) and illuminous intensity (L), i.e., ksub((1))[C 0 sup(Cl 2 ): 10 ppm] = 6.56 x 10 -5 L (L = 0 -- 1000 lx). Thus, the use of both sea-water addition and UV irradiation provides a probable method for decomposing a residual chlorine to the expected concentration. (author)

Management of NORM Residues

International Nuclear Information System (INIS)

2013-06-01

The IAEA attaches great importance to the dissemination of information that can assist Member States in the development, implementation, maintenance and continuous improvement of systems, programmes and activities that support the nuclear fuel cycle and nuclear applications, and that address the legacy of past practices and accidents. However, radioactive residues are found not only in nuclear fuel cycle activities, but also in a range of other industrial activities, including: - Mining and milling of metalliferous and non-metallic ores; - Production of non-nuclear fuels, including coal, oil and gas; - Extraction and purification of water (e.g. in the generation of geothermal energy, as drinking and industrial process water; in paper and pulp manufacturing processes); - Production of industrial minerals, including phosphate, clay and building materials; - Use of radionuclides, such as thorium, for properties other than their radioactivity. Naturally occurring radioactive material (NORM) may lead to exposures at some stage of these processes and in the use or reuse of products, residues or wastes. Several IAEA publications address NORM issues with a special focus on some of the more relevant industrial operations. This publication attempts to provide guidance on managing residues arising from different NORM type industries, and on pertinent residue management strategies and technologies, to help Member States gain perspectives on the management of NORM residues

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

Science.gov (United States)

Bueno, Amy N; Shrestha, Rashmi K; Ronau, Judith A; Babar, Aditya; Sheedlo, Michael J; Fuchs, Julian E; Paul, Lake N; Das, Chittaranjan

2015-10-06

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic

An Overview of the Prediction of Protein DNA-Binding Sites

Directory of Open Access Journals (Sweden)

Jingna Si

2015-03-01

Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Manual for implementing residual radioactivity guidelines

International Nuclear Information System (INIS)

Gilbert, T.L.; Eckerman, K.F.; Hansen, W.R.; Healy, J.W.; Kennedy, W.E.; Napier, B.A.; Solday, J.K.

1986-01-01

The US Department of Energy (DOE) has recently issued guidelines for residual radioactivity at Formerly Utilized Sites Remedial Action Program (FUSRAP) and remote Surplus Facilities Management Program (SFMP) sites. A manual for implementing these guidelines has been prepared jointly by four DOE laboratories (ANL, LANL, ORNL, and PNL) and is being issued as a supplement to the guidelines. The manual presents procedures and tables for deriving site-specific guidelines for levels of residual radionuclide concentrations in soil that must not be exceeded if a site is to be released for unrestricted use. Guidance for implementing DOE ALARA policy for remedial actions is also included. The concentration factor method is used in the pathway analysis for deriving soil guidelines. The analysis has been structured in a manner that explicitly identifies all of the factors involved. Tables are provided for dose-conversion factors and pathway factors from which environmental transport factors for each radionuclide and pathway may be calculated. The scenarios used for deriving the environmental transport factors and dose conversion factors, and the manner in which the information provided in the manual is used to derive site-specific soil guidelines will be presented

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase.

Science.gov (United States)

D'Ordine, Robert L; Linger, Rebecca S; Thai, Carolyn J; Davisson, V Jo

2012-07-24

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the

Measurement of residual CO2 saturation at a geological storage site using hydraulic tests

Science.gov (United States)

Rötting, T. S.; Martinez-Landa, L.; Carrera, J.; Russian, A.; Dentz, M.; Cubillo, B.

2012-12-01

Estimating long term capillary trapping of CO2 in aquifers remains a key challenge for CO2 storage. Zhang et al. (2011) proposed a combination of thermal, tracer, and hydraulic experiments to estimate the amount of CO2 trapped in the formation after a CO2 push and pull test. Of these three types of experiments, hydraulic tests are the simplest to perform and possibly the most informative. However, their potential has not yet been fully exploited. Here, a methodology is presented to interpret these tests and analyze which parameters can be estimated. Numerical and analytical solutions are used to simulate a continuous injection in a porous medium where residual CO2 has caused a reduction in hydraulic conductivity and an increase in storativity over a finite thickness (a few meters) skin around the injection well. The model results are interpreted using conventional pressure build-up and diagnostic plots (a plot of the drawdown s and the logarithmic derivative d s / d ln t of the drawdown as a function of time). The methodology is applied using the hydraulic parameters estimated for the Hontomin site (Northern Spain) where a Technology Demonstration Plant (TDP) for geological CO2 storage is planned to be set up. The reduction of hydraulic conductivity causes an increase in observed drawdowns, the increased storativity in the CO2 zone causes a delay in the drawdown curve with respect to the reference curve measured before CO2 injection. The duration (characteristic time) of these effects can be used to estimate the radius of the CO2 zone. The effects of reduced permeability and increased storativity are well separated from wellbore storage and natural formation responses, even if the CO2-brine interface is inclined (i.e. the CO2 forms a cone around the well). We find that both skin hydraulic conductivity and storativity (and thus residual CO2 saturation) can be obtained from the water injection test provided that water flow rate is carefully controlled and head build

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

Science.gov (United States)

Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

2014-01-01

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

Niagara Falls Storage Site environmental surveillance report for calendar year 1993

International Nuclear Information System (INIS)

1994-06-01

This report summarizes the results of environmental surveillance activities conducted at the Niagara Falls Storage Site (NFSS) during calendar year 1993. It includes an overview of site operations, the basis for radiological and nonradiological monitoring, a summary of the results, and the estimated dose to the offsite population. Environmental surveillance activities were conducted in accordance with the site environmental monitoring plan, which describes the rationale and design criteria for the surveillance program, the frequency of sampling and analysis, specific sampling and analysis procedures, and quality assurance requirements. NFSS is in compliance with National Emission Standards for Hazardous Air Pollutants (NESHAPs) Subpart H of the Clean Air Act as well as the requirements of the National Pollutant Discharge Elimination System (NPDES) under the Clean Water Act. Located in northwestern New York, the site covers 191 acres. From 1944 to the present, the primary use of NFSS has been storage of radioactive residues that were by-products of uranium production. Most onsite areas of residual radioactivity above regulatory guidelines were remediated during the early 1980s. Additional isolated areas of onsite contamination were remediated in 1989, and the materials were consolidated into the waste containment structure in 1991. Remediation of the site has now been completed

Ventricular activity cancellation in electrograms during atrial fibrillation with constraints on residuals' power.

Science.gov (United States)

Corino, Valentina D A; Rivolta, Massimo W; Sassi, Roberto; Lombardi, Federico; Mainardi, Luca T

2013-12-01

During atrial fibrillation (AF), cancellation of ventricular activity from atrial electrograms (AEG) is commonly performed by template matching and subtraction (TMS): a running template, built in correspondence of QRSs, is subtracted from the AEG to uncover atrial activity (AA). However, TMS can produce poor cancellation, leaving high-power residues. In this study, we propose to modulate the templates before subtraction, in order to make the residuals as similar as possible to the nearby atrial activity, avoiding high-power ones. The coefficients used to modulate the template are estimated by maximizing, via Multi-swarm Particle Swarm Optimization, a fitness function. The modulated TMS method (mTMS) was tested on synthetic and real AEGs. Cancellation performances were assessed using: normalized mean squared error (NMSE, computed on simulated data only), reduction of ventricular activity (VDR), and percentage of segments (PP) whose power was outside the standard range of the atrial power. All testings suggested that mTMS is an improvement over TMS alone, being, on simulated data, NMSE and PP significantly decreased while VDR significantly increased. Similar results were obtained on real electrograms (median values of CS1 recordings PP: 2.44 vs. 0.38 p < 0.001; VDR: 6.71 vs. 8.15 p < 0.001). Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

Managing Siting Activities for Nuclear Power Plants

International Nuclear Information System (INIS)

2012-01-01

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

Managing Siting Activities for Nuclear Power Plants

Energy Technology Data Exchange (ETDEWEB)

NONE

2012-06-15

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

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

International Nuclear Information System (INIS)

Miziorko, H.M.; Behnke, C.E.

1985-01-01

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

Energy Technology Data Exchange (ETDEWEB)

Sode, Olaseni; Voth, Gregory A., E-mail: gavoth@uchicago.edu [Department of Chemistry, James Franck Institute, Institute for Biophysical Dynamics, Computation Institute, The University of Chicago, Chicago, Illinois 60637, USA and Computing, Environment and Life Sciences, Argonne National Laboratory, Argonne, Illinois 60439 (United States)

2014-12-14

Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A{sup 2−}, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A{sup 2-} by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

International Nuclear Information System (INIS)

Sode, Olaseni; Voth, Gregory A.

2014-01-01

Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A 2− , a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A 2- by closing a cavity that could otherwise fill with water near the proximal Fe of the active site

Relocation of radioactive residuals store: environment effects statement

International Nuclear Information System (INIS)

1984-11-01

This Environment Effects Statement describes and assesses the likely environmental effects of the proposal to relocate the Health Commission's existing radioactive residuals store to a site within the established Dutson Downs waste disposal area, located 20 km south-east of Sale and 225 km east of Melbourne. The information presented demonstrates that the siting and construction of the proposed radioactive residuals store and the procedures to be adopted for the handling and storage of materials will not present an unacceptable risk to public health and safety, nor will it involve any significant adverse environmental effects

2010 Annual Summary Report for the Area 3 and Area 5 Radioactive Management Sites at the Nevada National Security Site, Nye County, Nevada

Energy Technology Data Exchange (ETDEWEB)

NSTec Environmental Management

2011-03-01

The U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office performed an annual review of the Area 3 and Area 5 Radioactive Waste Management Site (RWMS) Performance Assessments (PAs) and Composite Analyses (CAs) in fiscal year (FY) 2010. This annual summary report presents data and conclusions from the FY 2010 review, and determines the adequacy of the PAs and CAs. Operational factors (e.g., waste forms and containers, facility design, and waste receipts), closure plans, monitoring results, and research and development (R&D) activities were reviewed to determine the adequacy of the PAs. Likewise, the environmental restoration activities at the Nevada National Security Site (NNSS) (formerly the Nevada Test Site) relevant to the sources of residual radioactive material that are considered in the CAs, the land-use planning, and the results of the environmental monitoring and R&D activities were reviewed to determine the adequacy of the CAs.

Direct instrumental identification of catalytically active surface sites

Science.gov (United States)

Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

2017-09-01

The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

Crystal structures reveal metal-binding plasticity at the metallo-β-lactamase active site of PqqB from Pseudomonas putida

Energy Technology Data Exchange (ETDEWEB)

Tu, Xiongying; Latham, John A.; Klema, Valerie J.; Evans III, Robert L.; Li, Chao; Klinman, Judith P.; Wilmot, Carrie M. (UMM); (UCB)

2017-08-19

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.

Using cotton plant residue to produce briquettes

Energy Technology Data Exchange (ETDEWEB)

Coates, W. [University of Arizona, Tucson, AZ (United States). Bioresources Research Facility

2000-07-01

In Arizona, cotton (Gossypium) plant residue left in the field following harvest must be buried to prevent it from serving as an overwintering site for insects such as the pink bollworm. Most tillage operations employed to incorporate the residue into the soil are energy intensive and often degrade soil structure. Trials showed that cotton plant residue could be incorporated with pecan shells to produce commercially acceptable briquettes. Pecan shell briquettes containing cotton residue rather than waste paper were slightly less durable, when made using equivalent weight mixtures and moisture contents. Proximate and ultimate analyses showed the only difference among briquette samples to be a higher ash content in those made using cotton plant residue. Briquettes made with paper demonstrated longer flame out time, and lower ash percentage, compared to those made with cotton plant residue. (author)

Identification of the Allosteric Regulatory Site of Insulysin

Energy Technology Data Exchange (ETDEWEB)

Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W. (U. Sao Paulo); (Kentucky)

2012-05-25

Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the A{beta} peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

Identification of the Allosteric Regulatory Site of Insulysin

Energy Technology Data Exchange (ETDEWEB)

Noinaj, Nicholas; Bhasin, Sonia K.; Song, Eun Suk; Scoggin, Kirsten E.; Juliano, Maria A.; Juliano, Luiz; Hersh, Louis B.; Rodgers, David W.; Gerrard, Juliet Ann

2011-06-24

Background Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer's disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP. Principal Findings The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits. Conclusions/Significance Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.

Remaining Sites Verification Package for the 100-D-9 Boiler Fuel Oil Tank Site. Attachment to Waste Site Reclassification Form 2006-030

International Nuclear Information System (INIS)

Dittmer, L.M.

2006-01-01

The 100-D-9 site is the former location of an underground storage tank used for holding fuel for the 184-DA Boiler House. Results of soil-gas samples taken from six soil-gas probes in a rectangle around the site the tank had been removed from concluded that there were no volatile organic compounds at detectable levels in the area. The 100-D-9 Boiler Fuel Oil Tank Site meets the remedial action objectives specified in the Remaining Sites ROD. The results demonstrated that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River

Active sites environmental monitoring Program - Program Plan: Revision 2

International Nuclear Information System (INIS)

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

1994-05-01

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

Removal of residual nuclei following a cavitation event using low-amplitude ultrasound.

Science.gov (United States)

Duryea, Alexander P; Cain, Charles A; Tamaddoni, Hedieh A; Roberts, William W; Hall, Timothy L

2014-10-01

Microscopic residual bubble nuclei can persist on the order of 1 s following a cavitation event. These bubbles can limit the efficacy of ultrasound therapies such as shock wave lithotripsy and histotripsy, because they attenuate pulses that arrive subsequent to their formation and seed repetitive cavitation activity at a discrete set of sites (cavitation memory). Here, we explore a strategy for the removal of these residual bubbles following a cavitation event, using low-amplitude ultrasound pulses to stimulate bubble coalescence. All experiments were conducted in degassed water and monitored using high-speed photography. In each case, a 2-MHz histotripsy transducer was used to initiate cavitation activity (a cavitational bubble cloud), the collapse of which generated a population of residual bubble nuclei. This residual nuclei population was then sonicated using a 1 ms pulse from a separate 500-kHz transducer, which we term the bubble removal pulse. Bubble removal pulse amplitudes ranging from 0 to 1.7 MPa were tested, and the backlit area of shadow from bubbles remaining in the field following bubble removal was calculated to quantify efficacy. It was found that an ideal amplitude range exists (roughly 180 to 570 kPa) in which bubble removal pulses stimulate the aggregation and subsequent coalescence of residual bubble nuclei, effectively removing them from the field. Further optimization of bubble removal pulse sequences stands to provide an adjunct to cavitation-based ultrasound therapies such as shock wave lithotripsy and histotripsy, mitigating the effects of residual bubble nuclei that currently limit their efficacy.

A conserved residue of l-alanine dehydrogenase from Bacillus pseudofirmus, Lys-73, participates in the catalytic reaction through hydrogen bonding.

Science.gov (United States)

He, Guangzheng; Xu, Shujing; Wang, Shanshan; Zhang, Qing; Liu, Dong; Chen, Yuling; Ju, Jiansong; Zhao, Baohua

2018-03-01

A multiple protein sequence alignment of l-alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of l-alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coli, purified by His 6 -tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to l-alanine (15Umg -1 ) with an optimal temperature and pH of 40°C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in k cat /K m towards l-alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75. Copyright © 2017 Elsevier Inc. All rights reserved.

Minimization of zirconium chlorinator residues

International Nuclear Information System (INIS)

Green, G.K.; Harbuck, D.D.

1995-01-01

Zirconium chlorinator residues contain an array of rare earths, scandium, unreacted coke, and radioactive thorium and radium. Because of the radioactivity, the residues must be disposed in special waste containment facilities. As these sites become more congested, and with stricter environmental regulations, disposal of large volumes of wastes may become more difficult. To reduce the mass of disposed material, the US Bureau of Mines (USBM) developed technology to recover rare earths, thorium and radium, and unreacted coke from these residues. This technology employs an HCl leach to solubilize over 99% of the scandium and thorium, and over 90% of the rare earths. The leach liquor is processed through several solvent extraction stages to selectively recover scandium, thorium, and rare earths. The leach residue is further leached with an organic acid to solubilize radium, thus allowing unreacted coke to be recycled to the chlorinator. The thorium and radium waste products, which comprise only 2.1% of the original residue mass, can then be sent to the radioactive waste facility

Measuring site occupancy

DEFF Research Database (Denmark)

Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James

2014-01-01

occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass...... peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise...... and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response....

Evaluation of residual activity of solid waste generated in nuclear medicine services of Porto Alegre - Brazil

International Nuclear Information System (INIS)

Xavier, Ana M.; Alabarse, Frederico Gil; Magalhaes, Maisa Haiidamus; Guerrero, Jesus Salvador Perez

2008-01-01

An experimental programme to estimate, with a better degree of accuracy, the activity that remains adsorbed in flasks and syringes used in Nuclear Medicine Services for the administration of radionuclides to patients submitted to diagnostic or therapy was conducted under the coordination of the Radioactive Waste Division of the Brazilian Nuclear Energy Commission. The adopted recommendation in Brazil to allow an expedite solid waste management in nuclear medicine facilities, up to the present, is to consider that 2% of the initial activity remains adsorbed in the solid waste, which easily allows the calculation of the storage time to achieve regulatory clearance levels by decay. This research evaluates 17 different kinds of radio pharmaceuticals and three radioisotopes: 99m Tc, 67 Ga and 201 Tl. Results obtained by means of a weighting method to estimate the residual mass in flasks show that the ratio of the mass of the liquid that remains in the solid waste to the mass of the empty flask is constant. This suggests that the residual activity depends on the initial activity concentration of radiopharmaceutical contained in each flask, as assumed by the regulatory body. Additionally, results obtained by determining the remaining activity in flasks, shortly after the injection of its radionuclides contents in patients, indicate that an average value for the residual activity of the order of 10% of the initial activity contained in the flasks or syringes can be adopted instead of the previously assumed 2%. It is suggested that the more conservative average value obtained in the present work for the activity that remains in flasks and syringes, that is, 10% of the initial activity, could be adopted to determine the decay storage time before the release of solid waste in the urban conventional land fill disposal system. (author)

Biotransformation of fluoroquinolone antibiotics by ligninolytic fungi - Metabolites, enzymes and residual antibacterial activity

Czech Academy of Sciences Publication Activity Database

Čvančarová, Monika; Moeder, M.; Filipová, Alena; Cajthaml, Tomáš

2015-01-01

Roč. 136, OCT 2015 (2015), s. 311-320 ISSN 0045-6535 R&D Projects: GA TA ČR TE01020218; GA ČR GA13-28283S Institutional support: RVO:61388971 Keywords : Fluoroquinolone antibiotics * White rot fungi * Residual antibacterial activity Subject RIV: EE - Microbiology, Virology Impact factor: 3.698, year: 2015

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Energy Technology Data Exchange (ETDEWEB)

Pochapsky, Thomas C., E-mail: pochapsk@brandeis.edu; Pochapsky, Susan S.; Ju Tingting [Brandeis University, Department of Chemistry (United States); Hoefler, Chris [Brandeis University, Department of Biochemistry (United States); Liang Jue [Brandeis University, Department of Chemistry (United States)

2006-02-15

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O{sub 2}) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni{sup 2+}-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.

A Quantitative Tool to Distinguish Isobaric Leucine and Isoleucine Residues for Mass Spectrometry-Based De Novo Monoclonal Antibody Sequencing

Science.gov (United States)

Poston, Chloe N.; Higgs, Richard E.; You, Jinsam; Gelfanova, Valentina; Hale, John E.; Knierman, Michael D.; Siegel, Robert; Gutierrez, Jesus A.

2014-07-01

De novo sequencing by mass spectrometry (MS) allows for the determination of the complete amino acid (AA) sequence of a given protein based on the mass difference of detected ions from MS/MS fragmentation spectra. The technique relies on obtaining specific masses that can be attributed to characteristic theoretical masses of AAs. A major limitation of de novo sequencing by MS is the inability to distinguish between the isobaric residues leucine (Leu) and isoleucine (Ile). Incorrect identification of Ile as Leu or vice versa often results in loss of activity in recombinant antibodies. This functional ambiguity is commonly resolved with costly and time-consuming AA mutation and peptide sequencing experiments. Here, we describe a set of orthogonal biochemical protocols, which experimentally determine the identity of Ile or Leu residues in monoclonal antibodies (mAb) based on the selectivity that leucine aminopeptidase shows for n-terminal Leu residues and the cleavage preference for Leu by chymotrypsin. The resulting observations are combined with germline frequencies and incorporated into a logistic regression model, called Predictor for Xle Sites (PXleS) to provide a statistical likelihood for the identity of Leu at an ambiguous site. We demonstrate that PXleS can generate a probability for an Xle site in mAbs with 96% accuracy. The implementation of PXleS precludes the expression of several possible sequences and, therefore, reduces the overall time and resources required to go from spectra generation to a biologically active sequence for a mAb when an Ile or Leu residue is in question.

Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

Science.gov (United States)

Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

2018-01-20

PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Stabilization of plutonium bearing residues at Lawrence Livermore National Laboratory

International Nuclear Information System (INIS)

Bronson, M.C.; Van Konynenburg, R.A.; Ebbinghaus, B.B.

1995-01-01

The US Department of Energy's (US DOE) Lawrence Livermore National Laboratory (LLNL) has plutonium holdings including metal, oxide and residue materials, all of which need stabilization of some type. Residue materials include calcined ash, calcined precipitates, pyrochemical salts, glove box sweepings, metallurgical samples, graphite, and pyrochemical ceramic crucibles. These residues are typical of residues stored throughout the US DOE plutonium sites. The stabilization process selected for each of these residues requires data on chemical impurities, physical attributes, and chemical forms of the plutonium. This paper outlines the characterization and stabilization of LLNL ash residues, pyrochemical salts, and graphite

Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

Science.gov (United States)

Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

1995-08-01

A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507.

Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study

Science.gov (United States)

Linde, Dolores; Pogni, Rebecca; Cañellas, Marina; Lucas, Fátima; Guallar, Victor; Baratto, Maria Camilla; Sinicropi, Adalgisa; Sáez-Jiménez, Verónica; Coscolín, Cristina; Romero, Antonio; Medrano, Francisco Javier; Ruiz-Dueñas, Francisco J.; Martínez, Angel T.

2014-01-01

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant. PMID:25495127

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

Directory of Open Access Journals (Sweden)

Marlien Pieters

Full Text Available Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g, platelet-containing (352 g and platelet-rich plasma (200 g were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation. Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly

Allowable Residual Contamination Levels in soil for decommissioning the Shippingport Atomic Power Station site

International Nuclear Information System (INIS)

Kennedy, W.E. Jr.; Napier, B.A.; Soldat, J.K.

1983-09-01

As part of decommissioning the Shippingport Atomic Power Station, a fundamental concern is the determination of Allowable Residual Contamination Levels (ARCL) for radionuclides in the soil at the site. The ARCL method described in this report is based on a scenario/exposure-pathway analysis and compliance with an annual dose limit for unrestricted use of the land after decommissioning. In addition to naturally occurring radionuclides and fallout from weapons testing, soil contamination could potentially come from five other sources. These include operation of the Shippingport Station as a pressurized water reactor, operations of the Shippingport Station as a light-water breeder, operation of the nearby Beaver Valley reactors, releases during decommissioning, and operation of other nearby industries, including the Bruce-Mansfield coal-fired power plants. ARCL values are presented for 29 individual radionculides and a worksheet is provided so that ARCL values can be determined for any mixture of the individual radionuclides for any annual dose limit selected. In addition, a worksheet is provided for calculating present time soil concentration value that will decay to the ARCL values after any selected period of time, such as would occur during a period of restricted access. The ARCL results are presented for both unconfined (surface) and confined (subsurface) soil contamination. The ARCL method and results described in this report provide a flexible means of determining unrestricted-use site release conditions after decommissioning the Shippingport Atomic Power Station

Long-term surveillance plan for the Mexican Hat disposal site, Mexican Hat, Utah

International Nuclear Information System (INIS)

1996-01-01

This plan describes the long-term surveillance activities for the Uranium Mill Tailings Remedial Action (UMTRA) Project disposal site at Mexican Hat, Utah. The US Department of Energy (DOE) will carry out these activities to ensure that the disposal site continues to function as designed. This long-term surveillance plan (LTSP) was prepared as a requirement for acceptance under the US Nuclear Regulatory Commission (NRC) general license for custody and long-term care of residual radioactive material (RRM). This LTSPC documents the land ownership interests and details how the long-term care of the disposal site will be accomplished

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

Directory of Open Access Journals (Sweden)

Sivananthaperumal Shanmugasundararaj

Full Text Available Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

Promoter proximal polyadenylation sites reduce transcription activity

DEFF Research Database (Denmark)

Andersen, Pia Kjølhede; 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...

Energy transfer at the active sites of heme proteins

International Nuclear Information System (INIS)

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

1995-01-01

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

TANK FARM CLOSURE - A NEW TWIST ON REGULATORY STRATEGIES FOR CLOSURE OF WASTE TANK RESIDUALS FOLLOWING NUREG

International Nuclear Information System (INIS)

LEHMAN LL

2008-01-01

Waste from a number of single-shell tanks (SST) at the U.S. Department of Energy's (DOE) Hanford Site has been retrieved by CH2M HILL Hanford Group to fulfill the requirements of the 'Hanford Federal Facility Agreement and Consent Order (HFFACO) [1]. Laboratory analyses of the Hanford tank residual wastes have provided concentration data which will be used to determine waste classification and disposal options for tank residuals. The closure of tank farm facilities remains one of the most challenging activities faced by the DOE. This is due in part to the complicated regulatory structures that have developed. These regulatory structures are different at each of the DOE sites, making it difficult to apply lessons learned from one site to the next. During the past two years with the passage of the Section 3116 of the 'Ronald Reagan Defense Authorization Act of 2005' (NDAA) [2] some standardization has emerged for Savannah River Site and the Idaho National Laboratory tank residuals. Recently, with the issuance of 'NRC Staff Guidance for Activities Related to US. Department of Energy Waste Determinations' (NUREG-1854) [3] more explicit options may be considered for Hanford tank residuals than are presently available under DOE Orders. NUREG-1854, issued in August 2007, contains several key pieces of information that if utilized by the DOE in the tank closure process, could simplify waste classification and streamline the NRC review process by providing information to the NRC in their preferred format. Other provisions of this NUREG allow different methods to be applied in determining when waste retrieval is complete by incorporating actual project costs and health risks into the calculation of 'technically and economically practical'. Additionally, the NUREG requires a strong understanding of the uncertainties of the analyses, which given the desire of some NRC/DOE staff may increase the likelihood of using probabilistic approaches to uncertainty analysis. The purpose

Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

DEFF Research Database (Denmark)

Sulewska, Anna Maria

ABSTRACT Activity of endogenous enzymes may cause browning of fruits and vegetables. These enzymes can be inactivated, for example by heat treatment, but the response of enzymes to heat treatment depends on many factors. Foods are very complex systems and the stability of enzymes......-casein on the enzymatic activity of three targets was tested by monitoring enzyme activity after heat treatment and by measuring the intensity of scattered light during and after heat treatment. β-Casein was shown to interact at elevated temperatures with three selected targets:horseradish peroxidase, tyrosinase from......, residual activity of horseradish peroxidase was lower in samples containing BSA than in samples without any addition. Horseradish peroxidase heated with BSA did not regain activity within one hour after treatment. BSA is often added to enzyme solutions to prevent enzyme adhesion to vial surfaces...

Developing ceramic based technology for the immobilisation of waste on the Sellafield site - 16049

International Nuclear Information System (INIS)

Scales, C.R.; Maddrell, E.R.; Dowson, Mark

2009-01-01

National Nuclear Laboratory, in collaboration with the Australian Nuclear Science and Technology Organisation, is developing hot isostatic press (HIP) based ceramic technology for the immobilisation of a diverse range of wastes arising from nuclear fuel processing activities on the Sellafield site. Wasteform compositions have been identified and validated for the immobilisation of these plutonium containing wastes and residues in glass-ceramic and ceramic forms. A full scale inactive facility has been constructed at NNL's Workington Laboratory to support the demonstration of the technology. Validation of the inactive wasteform development using plutonium has been carried out at ANSTO's Lucas Heights facility. A feasibility study has been conducted to evaluate the construction and operation of a plutonium active pilot facility which would demonstrate the immobilisation of actual residues in the NNL Central Lab. This could form the basis of a facility to treat the plutonium wastes and residues in their entirety. The technology is being explored for the immobilisation of additional wastes arising on the Sellafield site taking advantage of the investment already made in skills and facilities. (authors)