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Sample records for 15n backbone chemical

  1. Combining ambiguous chemical shift mapping with structure-based backbone and NOE assignment from 15N-NOESY

    Jang, Richard

    2011-01-01

    Chemical shift mapping is an important technique in NMRbased drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule\\'s introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically. However, automated methods are necessary for high-throughput drug screening. We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C- labeling, to resolve the ambiguities for a one-toone mapping. On the three proteins, it achieves an average accuracy of 94% or better. Copyright © 2011 ACM.

  2. Backbone and Ile-δ1, Leu, Val Methyl 1H, 13C and 15N NMR chemical shift assignments for human interferon-stimulated gene 15 protein

    Yin, Cuifeng; Aramini, James M.; Ma, LiChung; Cort, John R.; Swapna, G.V.T.; Krug, R. M.; Montelione, Gaetano

    2011-10-01

    Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins in a variety of ways. Here we report nearly complete backbone 1HN, 15N, 13CO, and 13Ca, as well as side chain 13Cb, methyl (Ile-d1, Leu, Val), amide (Asn, Gln), and indole NH (Trp) NMR resonance assignments for the 157-residue human ISG15 protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically important human ISG15 protein.

  3. Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone {sup 15}N or {sup 13}C′ chemical shifts of multiple contiguous residues in highly resolved 3D spectra

    Yoshimura, Yuichi; Kulminskaya, Natalia V.; Mulder, Frans A. A., E-mail: fmulder@chem.au.dk [Aarhus University, Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO) (Denmark)

    2015-02-15

    Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone {sup 1}H{sup N}, {sup 15}N{sup H}, and {sup 13}C′ resonance assignments to be completed from a single pair of 3D experiments.

  4. (H)N(COCA)NH and HN(COCA)NH experiments for 1H-15N backbone assignments in 13C/15N-labeled proteins

    Bracken, Clay; Palmer, Arthur G. III [Columbia University, Department of Biochemistry and Molecular Biophysics (United States); Cavanagh, John [New York State Department of Health, NMR Structural Biology Facility, Wadsworth Center (United States)

    1997-01-15

    Triple resonance HN(COCA)NH pulse sequences for correlating 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins that utilize overlapping coherence transfer periods provide increased sensitivity relative to pulse sequences that utilize sequential coherence transfer periods. Although the overlapping sequence elements reduce the overall duration of the pulse sequences, the principal benefit derives from a reduction in the number of 180 deg. pulses. Two versions of the technique are presented: a 3D (H)N(COCA)NH experiment that correlates 15N(i),1H(i-1), and 15N(i-1) spins, and a 3D HN(COCA)NH experiment that correlates 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins by simultaneously encoding the 1H(i) and 15N(i) chemical shifts during the t1 evolution period. The methods are demonstrated on a 13C/15N-enriched sample of the protein ubiquitin and are easily adapted for application to 2H/13C/15N-enriched proteins.

  5. Combining automated peak tracking in SAR by NMR with structure-based backbone assignment from 15N-NOESY

    Jang, Richard

    2012-03-21

    Background: Chemical shift mapping is an important technique in NMR-based drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule\\'s introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically, which is not efficient for high-throughput drug screening.Results: We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C-labeling, to resolve the ambiguities for a one-to-one mapping. On the three proteins, it achieves an average accuracy of 94% or better.Conclusions: Our mathematical programming approach for modeling chemical shift mapping as a graph problem, while modeling the errors directly, is potentially a time- and cost-effective first step for high-throughput drug screening based on limited NMR data and homologous 3D structures. 2012 Jang et al.; licensee BioMed Central Ltd.

  6. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study.

    Sahu, S C; Bhuyan, A K; Udgaonkar, J B; Hosur, R V

    2000-10-01

    Backbone dynamics of uniformly 15N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 degrees C, pH 6.6, using 15N relaxation data obtained from proton-detected 2D [1H]-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear [1H]-15N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide 15N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (tau(m)) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motion's cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action. PMID

  7. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

    Backbone dynamics of uniformly 15N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 deg. C, pH 6.6, using 15N relaxation data obtained from proton-detected 2D {1H}-15N NMR spectroscopy. 15N spin-lattice relaxation rate constants (R1), spin-spin relaxation rate constants (R2), and steady-state heteronuclear {1H}-15N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide 15N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (τm) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action

  8. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

    Sahu, Sarata C. [Tata Institute of Fundamental Research, Department of Chemical Sciences (India); Bhuyan, Abani K.; Udgaonkar, Jayant B. [University of Agricultural Sciences (UAS), National Centre for Biological Sciences, Tata Institute of Fundamental Research (India); Hosur, R.V. [Tata Institute of Fundamental Research, Department of Chemical Sciences (India)

    2000-10-15

    Backbone dynamics of uniformly {sup 15}N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 deg. C, pH 6.6, using {sup 15}N relaxation data obtained from proton-detected 2D {l_brace}{sup 1}H{r_brace}-{sup 15}N NMR spectroscopy. {sup 15}N spin-lattice relaxation rate constants (R{sub 1}), spin-spin relaxation rate constants (R{sub 2}), and steady-state heteronuclear {l_brace}{sup 1}H{r_brace}-{sup 15}N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide {sup 15}N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times ({tau}{sub m}) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface

  9. Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

    Akke, M; Skelton, N J; Kördel, J; Palmer, A G; Chazin, W J

    1993-09-21

    The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site. PMID:8373781

  10. Backbone and sidechain 1H, 15N and 13C assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa

    Koveal, Dorothy; Jayasundera, Thusitha B.; Wood, Thomas K.; Peti, Wolfgang; Page, Rebecca

    2012-01-01

    The backbone and side chain resonance assignments of the Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa have been determined based on triple-resonance experiments using uniformly [13C,15N]-labeled protein. This assignment is the first step towards the determination of the 3-dimensional structure of TpbA.

  11. Predicting 15N chemical shifts in proteins using the preceding residue-specific individual shielding surfaces from φ, ψi-1, and χ1torsion angles

    Empirical shielding surfaces are most commonly used to predict chemical shifts in proteins from known backbone torsion angles, φ and ψ. However, the prediction of 15N chemical shifts using this technique is significantly poorer, compared to that for the other nuclei such as 1Hα, 13Cα, and 13Cβ. In this study, we investigated the effects from the preceding residue and the side-chain geometry, χ1, on 15N chemical shifts by statistical methods. For an amino acid sequence XY, the 15N chemical shift of Y is expressed as a function of the amino acid types of X and Y, as well as the backbone torsion angles, φ and ψi-1. Accordingly, 380 empirical 'Preceding Residue Specific Individual (PRSI)' 15N chemical shift shielding surfaces, representing all the combinations of X and Y (except for Y=Pro), were built and used to predict 15N chemical shift from φ and ψi-1. We further investigated the χ1 effects, which were found to account for differences in 15N chemical shifts by ∼5 ppm for amino acids Val, Ile, Thr, Phe, His, Tyr, and Trp. Taking the χ1 effects into account, the χ1-calibrated PRSI shielding surfaces (XPRSI) were built and used to predict 15N chemical shifts for these amino acids. We demonstrated that 15N chemical shift predictions are significantly improved by incorporating the preceding residue and χ1 effects. The present PRSI and XPRSI shielding surfaces were extensively compared with three recently published programs, SHIFTX (Neal et al., 2003), SHIFTS (Xu and Case, 2001 and 2002), and PROSHIFT (Meiler, 2003) on a set of ten randomly selected proteins. A set of Java programs using XPRSI shielding surfaces to predict 15N chemical shifts in proteins were developed and are freely available for academic users at http://www.pronmr.com or by sending email to one of the authors Yunjun Wang

  12. Variation of natural 15N abundance (δ15N) in greenhouse tomato and available nitrogen in soil supplied with cow manure or chemical fertilizers

    Cow manure or chemical fertilizers applied to greenhouse-grown tomato changed N contents and natural 15N abundance (δ15N) in tomato plants and the δ15N values of available N in soil. Cow manure increased and chemical fertilizers decreased the δ15N values of tomato plants. In the early periods of tomato culture with cow manure, the δ15N values of nitrate nitrogen of soil were higher than those of whole cow manure N, and, thereafter, dropped to δ15N values between those of soil and cow manure. Application of chemical fertilizers to soil immediately raised the δ15N values of ammonium nitrogen in soil but they dropped quickly to δ15N values between those of soil and fertilizers. On the estimation of the soil-derived N, manure-derived N and fertilizer-derived N in tomato plants based on the δ15N values of sources, much caution should be paid concerning the isotopic variation caused by N sources and isotopic fractionation during N transformation in soil. (author)

  13. Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

    Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in φ/ψ/χ1 torsion angles and sequence similarity to the query triplet of interest. The database contains 15N, 1HN, 1Hα, 13Cα, 13Cβ and 13C' chemical shifts for 200 proteins for which a high resolution X-ray (≤2.4 A) structure is available. The relative importance of the weighting factors for the φ/ψ/χ1 angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for 15N, 1HN, 1Hα, 13Cα, 13Cβ and 13C', respectively, including outliers

  14. CO{sub H}(N)CACB experiments for assigning backbone resonances in {sup 13}C/{sup 15}N-labeled proteins

    Astrof, Nathan; Bracken, Clay; Cavanagh, John; Palmer, Arthur G

    1998-05-15

    A triple resonance NMR experiment, denoted CO{sub H}(N)CACB, correlates{sup 1}H{sup N} and {sup 13}CO spins with the{sup 13}C{sup {alpha}} and{sup 13}C{sup {beta}} spins of adjacent amino acids. The pulse sequence is an 'out-and-back' design that starts with{sup 1}H{sup N} magnetization and transfers coherence via the {sup 15}N spin simultaneously to the {sup 13}CO and{sup 13}C{sup {alpha}} spins, followed by transfer to the{sup 13}C{sup {beta}} spin. Two versions of the sequence are presented: one in which the {sup 13}CO spins are frequency labeled during an incremented t{sub 1} evolution period prior to transfer of magnetization from the {sup 13}C{sup {alpha}} to the{sup 13}C{sup {beta}} resonances, and one in which the{sup 13}CO spins are frequency labeled in a constant-time manner during the coherence transfer to and from the{sup 13}C{sup {beta}} resonances. Because {sup 13}COand {sup 15}N chemical shifts are largely uncorrelated, the technique will be especially useful when degeneracy in the{sup 1}H{sup N}-{sup 15}N chemical shifts hinders resonance assignment. The CO{sub H}(N)CACB experiment is demonstrated using uniformly {sup 13}C/{sup 15}N-labeled ubiquitin.

  15. Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by {sup 15}N NMR relaxation methods

    Canales-Mayordomo, Angeles; Fayos, Rosa [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain); Angulo, Jesus; Ojeda, Rafael [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Martin-Pastor, Manuel [Unidad de RM y Unidad de RMN de Biomoleculas Asociada al CSIC, Laboratorio de Estructura e Estructura de Biomoleculas Jose Carracido (Spain); Nieto, Pedro M.; Martin-Lomas, Manuel [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain)], E-mail: jjbarbero@cib.csic.es

    2006-08-15

    The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

  16. Qualitative Study of Substituent Effects on NMR 15N and 17O Chemical Shifts

    Contreras, Rubén H.; Llorente, Tomás; Pagola, Gabriel I.; Bustamante, Manuel G.; Pasqualini, Enrique E.; Melo, Juan I.; Tormena, Cláudio F.

    2009-08-01

    A qualitative approach to analyze the electronic origin of substituent effects on the paramagnetic part of chemical shifts is described and applied to few model systems, where its potentiality can be appreciated. The formulation of this approach is based on the following grounds. The influence of different inter- or intramolecular interactions on a second-order property can be qualitatively predicted if it can be known how they affect the main virtual excitations entering into that second-order property. A set of consistent approximations are introduced in order to analyze the behavior of occupied and virtual orbitals that define some experimental trends of magnetic shielding constants. This approach is applied first to study the electronic origin of methyl-β substituent effects on both 15N and 17O chemical shifts, and afterward it is applied to a couple of examples of long-range substituent effects originated in charge transfer interactions such as the conjugative effect in aromatic compounds and σ-hyperconjugative interactions in saturated multicyclic compounds.

  17. Qualitative study of substituent effects on NMR (15)N and (17)O chemical shifts.

    Contreras, Rubén H; Llorente, Tomás; Pagola, Gabriel I; Bustamante, Manuel G; Pasqualini, Enrique E; Melo, Juan I; Tormena, Cláudio F

    2009-09-10

    A qualitative approach to analyze the electronic origin of substituent effects on the paramagnetic part of chemical shifts is described and applied to few model systems, where its potentiality can be appreciated. The formulation of this approach is based on the following grounds. The influence of different inter- or intramolecular interactions on a second-order property can be qualitatively predicted if it can be known how they affect the main virtual excitations entering into that second-order property. A set of consistent approximations are introduced in order to analyze the behavior of occupied and virtual orbitals that define some experimental trends of magnetic shielding constants. This approach is applied first to study the electronic origin of methyl-beta substituent effects on both (15)N and (17)O chemical shifts, and afterward it is applied to a couple of examples of long-range substituent effects originated in charge transfer interactions such as the conjugative effect in aromatic compounds and sigma-hyperconjugative interactions in saturated multicyclic compounds. PMID:19685922

  18. Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone {sup 13}CO-{sup 15}N rotational-echo double-resonance solid-state NMR

    Ghosh, Ujjayini; Xie Li; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

    2013-02-15

    The influenza virus fusion peptide is the N-terminal {approx}20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone {sup 13}CO-{sup 15}N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct {sup 13}C shifts.

  19. (1)H, (13)C, and (15)N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog.

    Moro, Sean L; Cocco, Melanie J

    2015-10-01

    The dinB homolog (Dbh) is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the (15)N, (1)H, and (13)C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains. PMID:26154586

  20. Separation of 15N by chemical exchange in NO, NO2 - HNO3 system under pressure

    The basic isotopic exchange reaction is responsible for the separation of 15N in the Nitrox system that between gaseous nitrogen oxides and aqueous nitric acid with a single stage separation factor α = 1.055 for 10M nitric acid, at 25 deg C and atmospheric pressure takes place. In order to know what happens in 15N separation at higher pressure, when the isotopic transport between two phases is improved, a stainless steel laboratory experimental plant with a 1000 mm long x 18 mm i.d. column, packed with triangular wire springs 1.8 x 1.8 x 0.2 mm2, was utilised. At 1.5 atm (absolute), and 2.36 ml x cm-2 x min-1 flow rate HETP was 7% smaller than at atmospheric pressure and 1.5 times smaller flow rate. HETP at 3.14 ml x cm-2 x min-1 flow rate and 1.8 atm is practically equal with that obtained at atmospheric pressure and 2 times smaller flow rate. The operation of the 15N separation plant at 1.8 atm (absolute), instead of atmospheric pressure, will permit doubling of the 10M nitric acid flow rate and of 15N production of the given column. (author)

  1. Inferential protein structure determination and refinement using fast, electronic structure based backbone amide chemical shift predictions

    Christensen, Anders S

    2015-01-01

    This report covers the development of a new, fast method for calculating the backbone amide proton chemical shifts in proteins. Through quantum chemical calculations, structure-based forudsiglese the chemical shift for amidprotonen in protein has been parameterized. The parameters are then implemented in a computer program called Padawan. The program has since been implemented in protein folding program Phaistos, wherein the method andvendes to de novo folding of the protein structures and to refine the existing protein structures.

  2. Automated prediction of 15N, 13Cα, 13Cβ and 13C' chemical shifts in proteins using a density functional database

    A database of peptide chemical shifts, computed at the density functional level, has been used to develop an algorithm for prediction of 15N and 13C shifts in proteins from their structure; the method is incorporated into a program called SHIFTS (version 4.0). The database was built from the calculated chemical shift patterns of 1335 peptides whose backbone torsion angles are limited to areas of the Ramachandran map around helical and sheet configurations. For each tripeptide in these regions of regular secondary structure (which constitute about 40% of residues in globular proteins) SHIFTS also consults the database for information about sidechain torsion angle effects for the residue of interest and for the preceding residue, and estimates hydrogen bonding effects through an empirical formula that is also based on density functional calculations on peptides. The program optionally searches for alternate side-chain torsion angles that could significantly improve agreement between calculated and observed shifts. The application of the program on 20 proteins shows good consistency with experimental data, with correlation coefficients of 0.92, 0.98, 0.99 and 0.90 and r.m.s. deviations of 1.94, 0.97, 1.05, and 1.08 ppm for 15N, 13Cα, 13Cβ and 13C', respectively. Reference shifts fit to protein data are in good agreement with 'random-coil' values derived from experimental measurements on peptides. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement

  3. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

    A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, ≥90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed (φ, ψ) torsion angles of ca 12º. TALOS-N also reports sidechain χ1 rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts

  4. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

    Shen Yang; Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2013-07-15

    A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, {>=}90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed ({phi}, {psi}) torsion angles of ca 12 Masculine-Ordinal-Indicator . TALOS-N also reports sidechain {chi}{sup 1} rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.

  5. Measurement of {sup 15}N relaxation in deuterated amide groups in proteins using direct nitrogen detection

    Vasos, Paul R.; Hall, Jennifer B. [University of Maryland, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization (United States); Kuemmerle, Rainer [Bruker Biospin AG, NMR Division (Switzerland); Fushman, David [University of Maryland, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization (United States)], E-mail: fushman@umd.edu

    2006-09-15

    {sup 15}N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of {sup 15}N chemical shielding can be obtained from {sup 15}N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from {sup 15}N-{sup 1}H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring {sup 15}N relaxation in deuterated amide groups in proteins, where the dipolar contribution to {sup 15}N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in {sup 15}N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse {sup 15}N relaxation for backbone amides in protein G in D{sub 2}O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional {sup 15}N relaxation measurements in H{sub 2}O. This analysis revealed a 17-24{sup o} angle between the NH-bond and the unique axis of the {sup 15}N chemical shielding tensor.

  6. Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection

    15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-1H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D2O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H2O. This analysis revealed a 17-24o angle between the NH-bond and the unique axis of the 15N chemical shielding tensor

  7. 1H, 13C and 13N chemical shifts and 1H-15N and 13C-15N heteronuclear spin-spin coupling constants n the NMR spectra of 5-substituted furfural oximes

    The 1H, 13C, and 15N NMR spectra of 15N-enriched 5-substituted furfural oximes were investigated. It was shown that the chemical shifts of the ring atoms and the oxime group correlate satisfactorily with the F and R substituent constants, whereas their sensitivity to the effect of the substituents is lower than in monosubstituted furan derivatives. The constants of spin-spin coupling between the ring protons and the oxime group were determined. An analysis of the 1H-1H spin-spin coupling constants (SSCC) on the basis of their stereospecificity indicates that the E isomers have primarily an s-trans conformation in polar dimethyl sulfoxide, whereas the Z isomers, on the other hand, have an s-cis conformation. The signs of the direct and geminal 13C-15N SSCC were determined for 5-trimethylsilylfurfural oxime

  8. Quantum chemical benchmark study on 46 RNA backbone families using a dinucleotide unit.

    Kruse, Holger; Mladek, Arnost; Gkionis, Konstantinos; Hansen, Andreas; Grimme, Stefan; Sponer, Jiri

    2015-10-13

    We have created a benchmark set of quantum chemical structure-energy data denoted as UpU46, which consists of 46 uracil dinucleotides (UpU), representing all known 46 RNA backbone conformational families. Penalty-function-based restrained optimizations with COSMO TPSS-D3/def2-TZVP ensure a balance between keeping the target conformation and geometry relaxation. The backbone geometries are close to the clustering-means of their respective RNA bioinformatics family classification. High-level wave function methods (DLPNO-CCSD(T) as reference) and a wide-range of dispersion-corrected or inclusive DFT methods (DFT-D3, VV10, LC-BOP-LRD, M06-2X, M11, and more) are used to evaluate the conformational energies. The results are compared to the Amber RNA bsc0χOL3 force field. Most dispersion-corrected DFT methods surpass the Amber force field significantly in accuracy and yield mean absolute deviations (MADs) for relative conformational energies of ∼0.4-0.6 kcal/mol. Double-hybrid density functionals represent the most accurate class of density functionals. Low-cost quantum chemical methods such as PM6-D3H+, HF-3c, DFTB3-D3, as well as small basis set calculations corrected for basis set superposition errors (BSSEs) by the gCP procedure are also tested. Unfortunately, the presently available low-cost methods are struggling to describe the UpU conformational energies with satisfactory accuracy. The UpU46 benchmark is an ideal test for benchmarking and development of fast methods to describe nucleic acids, including force fields. PMID:26574283

  9. Quantum Chemical Benchmark Study on 46 RNA Backbone Families Using a Dinucleotide Unit

    Kruse, H.; Mládek, Arnošt; Gkionis, Konstantinos; Hansen, A.; Grimme, S.; Šponer, Jiří

    2015-01-01

    Roč. 11, č. 10 (2015), s. 4972-4991. ISSN 1549-9618 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * DENSITY-FUNCTIONAL THEORY * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 5.498, year: 2014

  10. (1)H, (13)C, and (15)N chemical shift assignments of cyanobacteriochrome NpR6012g4 in the green-absorbing photoproduct state.

    Lim, Sunghyuk; Yu, Qinhong; Rockwell, Nathan C; Martin, Shelley S; Lagarias, J Clark; Ames, James B

    2016-04-01

    Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins with a tetrapyrrole (bilin) chromophore that belong to the phytochrome superfamily. Like phytochromes, CBCRs photoconvert between two photostates with distinct spectral properties. NpR6012g4 from Nostoc punctiforme is a model system for widespread CBCRs with conserved red/green photocycles. Atomic-level structural information for the photoproduct state in this subfamily is not known. Here, we report NMR backbone chemical shift assignments of the light-activated state of NpR6012g4 (BMRB no. 26577) as a first step toward determining its atomic resolution structure. PMID:26537963

  11. Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

    Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd, E-mail: reif@tum.d [Leibniz-Institut fuer Molekulare Pharmakologie (FMP) (Germany)

    2010-12-15

    We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline {alpha}-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual {sup 15}N-T{sub 1} timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s{sup -1}. Backbone amide {sup 15}N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41{epsilon}. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D{sub 2}O is employed as a solvent for sample preparation. Due to the intrinsically long {sup 15}N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

  12. Pseudo 5D HN(C)N Experiment to Facilitate the Assignment of Backbone Resonances in Proteins Exhibiting High Backbone Shift Degeneracy

    Kumar, Dinesh; Shukla, Vaibhav Kumar; Pandey, Himanshu; Arora, Ashish; Guleria, Anupam

    2014-01-01

    Assignment of protein backbone resonances is most routinely carried out using triple resonance three dimensional NMR experiments involving amide 1H and 15N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high degree of backbone shift degeneracy. In this backdrop, a novel reduced dimensionality (RD) experiment -(5,3)D-hNCO-CANH- is presented to facilitate (and/or to validate) the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information- leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The impr...

  13. Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

    Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

    2015-04-01

    Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W. PMID:24496608

  14. Pressure dependence of backbone chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

    Erlach, Markus Beck; Koehler, Joerg; Crusca, Edson; Kremer, Werner; Munte, Claudia E; Kalbitzer, Hans Robert

    2016-06-01

    For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms (1)H(α), (13)C(α) and (13)C' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The polynomial pressure coefficients B 1 and B 2 are dependent on the type of amino acid studied. The coefficients of a given nucleus show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure are also weakly correlated. PMID:27335085

  15. Combined solid state and solution NMR studies of {alpha},{epsilon}-{sup 15}N labeled bovine rhodopsin

    Werner, Karla; Lehner, Ines [Johann Wolfgang Goethe-Universitaet Frankfurt, Center for Biomolecular Magnetic Resonance (Germany); Dhiman, Harpreet Kaur [University of Pittsburgh School of Medicine, Department of Structural Biology (United States); Richter, Christian; Glaubitz, Clemens; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de; Klein-Seetharaman, Judith [Johann Wolfgang Goethe-Universitaet Frankfurt, Center for Biomolecular Magnetic Resonance (Germany); Khorana, H. Gobind [Massachusetts Institute of Technology, Departments of Biology and Chemistry (United States)], E-mail: khorana@mit.edu

    2007-04-15

    Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of {alpha},{epsilon}-{sup 15}N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state {sup 13}C,{sup 15}N-REDOR and HETCOR experiments of all possible {sup 13}C'{sub i-1} carbonyl/{sup 15}N{sub i}-tryptophan isotope labeled amide pairs, and H/D exchange {sup 1}H,{sup 15}N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone {sup 15}N nuclei and partially to their bound protons. {sup 1}H,{sup 15}N chemical shift assignment was achieved for indole side chains of Trp35{sup 1.30} and Trp175{sup 4.65}. {sup 15}N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175{sup 4.65} at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.

  16. Pseudo 5D HN(C)N experiment to facilitate the assignment of backbone resonances in proteins exhibiting high backbone shift degeneracy

    Kumar, Dinesh, E-mail: dineshcbmr@gmail.com [Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raibareli Road, Lucknow 226014 (India); Raikwal, Nisha [Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raibareli Road, Lucknow 226014 (India); Shukla, Vaibhav Kumar; Pandey, Himanshu; Arora, Ashish [Molecular and Structural Biology Division, CSIR, Central Drug Research Institute, Lucknow 226031 (India); Guleria, Anupam, E-mail: anuguleriaphy@gmail.com [Centre of Biomedical Research (CBMR), SGPGIMS Campus, Raibareli Road, Lucknow 226014 (India)

    2014-09-30

    Graphical abstract: - Highlights: • A reduced dimensionality experiment – referred as pseudo 5D HN(C)N- is presented. • Encodes highly resolved 5D spectral information in a 3D spectrum. • Superior in terms of peak dispersion. • Facilitates assignment of crowded HSQC spectra of moderately sized proteins. • Modulated {sup 15}N chemical shifts are used to break the amide shift degeneracy. - Abstract: Assignment of protein backbone resonances is most routinely carried out using triple resonance three-dimensional NMR experiments involving amide {sup 1}H/{sup 15}N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high-degree of backbone shift degeneracy. In this backdrop, a novel reduced-dimensionality (RD) experiment –(5, 3)D-hNCO-CANH- is presented to facilitate/validate the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide {sup 15}N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. H{sub i}N{sub i} and H{sub i−1}N{sub i−1}) for overlapping amide-NH peaks. The experiment -in combination with routine triple resonance 3D-NMR experiments involving backbone amide ({sup 1}H/{sup 15}N) and carbon ({sup 13}C{sup α}/{sup 13}C′) chemical shifts- will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner.

  17. Automated protein backbone assignment using the projection-decomposition approach

    Spectral projection experiments by NMR in conjunction with decomposition analysis have been previously introduced for the backbone assignment of proteins; various pulse sequences as well as the behaviour with low signal-to-noise or chemical shift degeneracy have been illustrated. As a guide for routine applications of this combined tool, we provide here a systematic analysis on different types of proteins using welldefined run-time parameters. As a second result of this study, the backbone assignment module SHABBA was extensively rewritten and improved. Calculations on ubiquitin yielded again fully correct and nearly complete backbone and CHβ assignments. For the 128 residue long azurin, missing assignments mostly affect Hα and Hβ. Among the remaining backbone (plus Cβ) nuclei 97.5% could be assigned with 1.0% differences to a reference. Finally, the new SHABBA algorithm was applied to projections recorded for a yeast histone protein domain at room temperature, where the protein is subject to partial unfolding: this leads to unobservable resonances (about a dozen missing signals in a normal 15N-HSQC) and extensive degeneracy among the resonances. From the clearly observable residues, 97.5% of the backbone and CHβresonances could be assigned, of which only 0.8 % showed differences to published shifts. An additional study on the protein MMP20, which exhibits spectral difficulties to an even larger extent, explores the limitations of the approach.

  18. (1)H, (15)N and (13)C chemical shift assignment of the Gram-positive conjugative transfer protein TraHpIP501.

    Fercher, Christian; Keller, Walter; Zangger, Klaus; Helge Meyer, N

    2016-04-01

    Conjugative transfer of DNA represents the most important transmission pathway in terms of antibiotic resistance and virulence gene dissemination among bacteria. TraH is a putative transfer protein of the type IV secretion system (T4SS) encoded by the Gram-positive (G+) conjugative plasmid pIP501. This molecular machine involves a multi-protein core complex spanning the bacterial envelope thereby serving as a macromolecular secretion channel. Here, we report the near complete (1)H, (13)C and (15)N resonance assignment of a soluble TraH variant comprising the C-terminal domain. PMID:26559076

  19. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S;

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  20. Evolution of the chemical (NH4) and isotopic (δ15N-NH4) composition of pig manure stored in an experimental deep pit

    during eight months, from November 2001 to July 2002 at atmospheric conditions. Ammonium concentration and its nitrogen isotopic composition were analysed weekly during the first six months and every ten days during the last two months. Ammonium concentration oscillates between 3000 and 4000 ppm, with no progressive decrease in time as it would be expected in a volatilisation process. This is caused by the loss of manure volume due to evaporation (50 % in eight months) which compensates the ammonia volatilisation and keeps the ammonium concentration stable. The nitrogen isotopic composition of the ammonium is controlled by the volatilisation process. During the first ten days, the pig manure has an isotopic composition between +8 and +10 per mille, and after eight months, it raises up to +25 per mille. However, the pig manure is not stored in the deep pits more than six months; consequently, the range of δ15NNH4 values to be considered for pig manure as an input of nitrogen contamination should be from +8 to +15 per mille. Still, this isotopic composition can be higher if manure is stored during the summer time since the higher temperatures may increase the volatilisation rate and raise the ammonium isotopic composition in a shorter period of time. Unlike synthetic fertilisers, which are characterised by a δ15N close to the 0 per mille, the organic fertilisers (pig manure) are enriched in 15N15N from +8 to +15 per mille). Therefore the nitrogen isotopic composition is a valuable tool to evaluate the origin of nitrate agricultural contaminations. As an example, nitrate nitrogen isotopic composition has been used in some areas in Catalonia to confirm that pig manure is the main contributor to the nitrate pollution in groundwaters

  1. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    Topham, Christopher M.; Smith, Jeremy C.

    2006-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the bindin...

  2. 15N analysis in nutritional and metabolic research of infancy

    Investigation of protein metabolism in nutritional pediatric research by means of 15N tracer techniques has been relatively seldom used up to now. 15N-labelled compounds for these purposes are not injurious to health. The technique is based on oral or intravenous application of the tracer substances and on 15N analysis of the urine fractions. The subsequent calculation of protein synthesis and breakdown rate, turnover, and the reutilisation of amino acids from protein breakdown as well as the size of the metabolic pool offers detailed information of protein metabolism. Determination of these parameters were performed in infants on breast milk, formula feeding and on chemically defined diet. As an example of utilisation of D-amino acids for protein synthesis the 15N-D-phenylalanine retention of parenteral nutrition was found to be 33% of the applied dosis at an average. An oral 15N-glycine loading test proved to be of value for the prediction of the therapeutic effect of human growth hormone. 15N tracer technique was also tested in utilizing 15N-urea for bacterial protein synthesis of the intestinal flora and by incorporation of 15N from 15N-glycine and 15N-lysine into the jejunal mucosa for measuring the enterocyte regeneration. (author)

  3. Methods of 15N tracer research in biological systems

    The application of the stable isotope 15N is of increasing importance in different scientific disciplines, especially in medicine, agriculture, and the biosciences. The close correlation between the growing interest and improvements of analytical procedures resulted in remarkable advances in the 15N tracer technique. On the basis of the latest results of 15N tracer research in life sciences and agriculture methods of 15N tracer research in biological systems are compiled. The 15N methodology is considered under three headings: Chemical analysis with a description of methods of sample preparation (including different separation and isolation methods for N-containing substances of biological and agricultural origin) and special procedures converting ammonia to molecular nitrogen. Isotopic analysis with a review on the most important methods of isotopic analysis of nitrogen: mass spectrometry (including the GC-MS technique), emission spectrometry, NMR spectroscopy, and other analytical procedures. 15N-tracer techniques with a consideration of the role of the isotope dilution analysis as well as different labelling techniques and the mathematical interpretation of tracer data (modelling, N turnover experiments). In these chapters also sources of errors in chemical and isotopic analysis, the accuracy of the different methods and its importance on tracer experiments are discussed. Procedures for micro scale 15N analysis and aspects of 15N analysis on the level of natural abundance are considered. Furthermore some remarks on isotope effects in 15N tracer experiments are made. (author)

  4. Synthesis of 15N labeled glyphosate

    Amongst the actually commercialized herbicides the Glyphosate is the most used in Brazil. Its efficiency as well as the others herbicides against undesirable weeds is harmed by its final composts left at the environment. Although studies has being carried out to improve the knowledge about the herbicides behavior at the environment its complexity has led them towards innumerous to new significant research work where the use of radiolabeled composts (radiative tracers) are recommended to evaluate their bio-availability in the soil. However is the use, the manipulation and the storage of radiolabeled composts is requires an extra care under chemical safety point of view. The use of non radiolabeled composts is a world tendency especially for field researches. Under this context the presented work describes a method for the synthesis of 15N labeled glyphosate. The 15N-herbicide was undertaken by phosphometilation with the phosphit dialquil and 15N-glycine. The tests where carried out through a micro scale production plant and of equimolars amounts. At these conditions it's was possible to reach approximately a 20% of yield. At the conclusion of a best operational condition its expected to offer another important toll that shall be used in glyphosate behavior at the environment and undesirably weeds. (author)

  5. Testing Backbone.js

    Roemer, Ryan

    2013-01-01

    This book is packed with the step by step tutorial and instructions in recipe format helping you setup test infrastructure and gradually advance your skills to plan, develop, and test your backbone applications.If you are a JavaScript developer looking for recipes to create and implement test support for your backbone application, then this book is ideal for you.

  6. NMR spectroscopic studies of 15N labelled geminally disubstituted cyclotriphosphazenes

    It is demonstrated by means of some selected 15N labelled geminally disubstituted cyclotriphosphazenes, 15N3P3X4Y2 (X = Cl; Y = F, NH2, or SEt), as an example, that the coupling constants 1Jsub(PN) may be of different signs. The absolute value of 1Jsub(PN) is significantly influenced only by those substituents, which are bonded to the phosphorus nucleus directly concerned in the coupling. Also the 15N chemical shifts are only changed by substituents on directly bonded phosphorus atoms. (author)

  7. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    Smith, Jeremy C [ORNL; Topham, Christopher [University of Heidelberg

    2007-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like base pair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNADNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.

  8. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form.

    Topham, Christopher M; Smith, Jeremy C

    2007-02-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA.DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

  9. Changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: An 15N NMR study

    The 15N-enriched ferrocytochrome c2from Rhodospirillum rubrum was studied by 15N NMR at different solvent pH values. The mobility and chemical shift to the N-terminal glutamic acid (335.4 ppm at pH 5.1) were found to depend on pH. It was least mobile between pH 8 and 9.0, which is explained in terms of pH-dependent conformational changes and formation of salt linkages and/or hydrogen bonds. The resonances of the lysine side chains are centered around 341.7 ppm at low pH and move upfield with pH by about 8.4 ppm with pH/sub a/ values of 10.8. The exchange rates of the εNH protons are lowest near the pK/sub a/ values. The protein is very stable in the pH range between 4.9 and 10.0 but unfolds abruptly at pH 10.5-11. Denaturation was verified by the measurement of several parameters by NMR. The renaturation of the protein demonstrates that the folding begins with reformation of home coordination and establishment of a hydrophobic core, followed by positioning of side chains and peptide backbones linking the nucleation centers. The repositioning processes had time scales of minutes to hours in contrast to the reported values of seconds in some studies

  10. Reduced Dimensionality (4,3)D-hnCOCANH Experiment: An Efficient Backbone Assignment tool for NMR studies of Proteins

    Kumar, Dinesh

    2013-01-01

    Sequence specific resonance assignment and secondary structure determination of proteins form the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (1H, 15N, 13Ca and 13C') resonances and secondary structure determination of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality (RD) experiment -(4,3)D-hnCOCANH and exploits the linear combinations of backbone (13Ca and 13C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text) for efficient and rapid data analysis. Further, the experiment leads to the spectrum with direct distinction of self (intra-residue) and sequential (inter-residue) carbon correlation peaks; these appear opposite in signs and therefore can easily be discriminated without using an additional complementary experiment. On ...

  11. Synthesis of {sup 15}N labeled glyphosate; Sintese do glifosato enriquecido com {sup 15}N

    Oliveira, Claudineia R. de; Bendassolli, Jose Albertino; Tavares, Glauco Arnold; Rossete, Alexssandra L.R.M.; Tagliassachi, Romulo Barbieri; Prestes, Cleuber Vieira [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Dept. de Isotopos Estaveis]. E-mail: crolivei@cena.usp.br

    2005-07-01

    Amongst the actually commercialized herbicides the Glyphosate is the most used in Brazil. Its efficiency as well as the others herbicides against undesirable weeds is harmed by its final composts left at the environment. Although studies has being carried out to improve the knowledge about the herbicides behavior at the environment its complexity has led them towards innumerous to new significant research work where the use of radiolabeled composts (radiative tracers) are recommended to evaluate their bio-availability in the soil. However is the use, the manipulation and the storage of radiolabeled composts is requires an extra care under chemical safety point of view. The use of non radiolabeled composts is a world tendency especially for field researches. Under this context the presented work describes a method for the synthesis of {sup 15}N labeled glyphosate. The {sup 15}N-herbicide was undertaken by phosphometilation with the phosphit dialquil and {sup 15}N-glycine. The tests where carried out through a micro scale production plant and of equimolars amounts. At these conditions it's was possible to reach approximately a 20% of yield. At the conclusion of a best operational condition its expected to offer another important toll that shall be used in glyphosate behavior at the environment and undesirably weeds. (author)

  12. Backbone amide linker strategy

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    In the backbone amide linker (BAL) strategy, the peptide is anchored not at the C-terminus but through a backbone amide, which leaves the C-terminal available for various modifications. This is thus a very general strategy for the introduction of C-terminal modifications. The BAL strategy was...... assemble the final peptide. One useful application of this strategy is in the synthesis of C-terminal peptide aldehydes. The C-terminal aldehyde is masked as an acetal during synthesis and then conveniently demasked in the final cleavage step to generate the free aldehyde. Another application is in the...

  13. Complete assignment of 1H, 13C and 15N chemical shifts for bovine β-lactoglobulin: Secondary structure and topology of the native state is retained in a partially unfolded form

    Although β-lactoglobulin (β-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine β-LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of β-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a13 C,15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric β-LG A. It includes eight antiparallel β-strands arranged in a barrel, flanked by an α-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth β-strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of β-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants

  14. Synthesis of 15 N double labelled urea

    Synthesis of double 15 N labelled urea by reacting 15 N - ammonia with elemental sulfur and carbon monoxide in a pressure vessel is presented. 15 NH3 was produced by H15 NO3 reduction with Dewarda alloy in alkaline solution, or by nitric monoxide reduction with hydrogen on metallic manganese. An average yield of 85% tacking into account 15 N - urea and 15 N ammonium sulfate, produced in the same time, and 99% urea purity (checked by I.R. spectroscopy and mass spectrometry) were obtained. (authors)

  15. Simultaneous acquisition of {sup 13}C{sup {alpha}}-{sup 15}N and {sup 1}H-{sup 15}N-{sup 15}N sequential correlations in proteins: application of dual receivers in 3D HNN

    Chakraborty, Swagata; Paul, Subhradip; Hosur, Ramakrishna V., E-mail: hosur@tifr.res.in [Tata Institute of Fundamental Research, Department of Chemical Sciences (India)

    2012-01-15

    We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable {sup 13}C{sup {alpha}} transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D {sup 13}C{sup {alpha}}-{sup 15}N sequential correlations along with the standard 3D {sup 15}N-{sup 15}N-{sup 1}H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.

  16. Complete resonance assignment for the polypeptide backbone of interleukin 1β using three-dimensional heteronuclear NMR spectroscopy

    The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1β has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. The authors show that the problems of amide NH and CαH chemical shift degeneracy that are prevalent for proteins of the size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1β. The complete list of 15N and 1H assignments is given for all the amide NH and CαH resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1β

  17. Geometry motivated alternative view on local protein backbone structures

    Zacharias, Jan; Knapp, Ernst Walter

    2013-01-01

    We present an alternative to the classical Ramachandran plot (R-plot) to display local protein backbone structure. Instead of the (ϕ, ψ)-backbone angles relating to the chemical architecture of polypeptides generic helical parameters are used. These are the rotation or twist angle ϑ and the helical rise parameter d. Plots with these parameters provide a different view on the nature of local protein backbone structures. It allows to display the local structures in polar (d, ϑ)-coordinates, whi...

  18. The DNA and RNA sugar-phosphate backbone emerges as the key player. An overview of quantum-chemical, structural biology and simulation studies

    Šponer, Jiří; Mládek, Arnošt; Šponer, Judit E.; Svozil, Daniel; Zgarbová, M.; Banáš, Pavel; Jurečka, P.; Otyepka, M.

    2012-01-01

    Roč. 14, č. 44 (2012), s. 15257-15277. ISSN 1463-9076 R&D Projects: GA ČR(CZ) GD203/09/H046; GA ČR(CZ) GAP208/10/2302; GA ČR(CZ) GAP208/11/1822; GA ČR(CZ) GAP208/12/1878; GA ČR(CZ) GA203/09/1476; GA ČR(CZ) GBP305/12/G034 Institutional research plan: CEZ:AV0Z50040702 Keywords : DNA * RNA * sugar -phosphate backbone Subject RIV: BO - Biophysics Impact factor: 3.829, year: 2012

  19. Resolution of the 15N balance enigma?

    The enigma of soil nitrogen balance sheets has been discussed for over 40 years. Many reasons have been considered for the incomplete recovery of 15N applied to soils, including sampling uncertainty, gaseous N losses from plants, and entrapment of soil gases. The entrapment of soil gases has been well documented for rice paddy and marshy soils but little or no work appears to have been done to determine entrapment in drained pasture soils. In this study 15N-labelled nitrate was applied to a soil core in a gas-tight glovebox. Water was applied, inducing drainage, which was immediately collected. Dinitrogen and N-2 were determined in the flux through the soil surface, and in the gases released into the glovebox as a result of irrigation or physical destruction of the core. Other components of the N balance were also measured, including soil inorganic-N and organic-N. Quantitative recovery of the applied 15N was achieved when the experiment was terminated 484 h after the 15N-labelled material was applied. Nearly 23% of the 15N was recovered in the glovebox atmosphere as N2 and N2O due to diffusion from the base of the soil core, convective flow after irrigation, and destructive soil sampling. This 15N would normally be unaccounted for using the sampling methodology typically employed in 15N recovery experiments. Copyright (2001) CSIRO Publishing

  20. Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

    Ovchinnikova, Tatyana V; Shenkarev, Zakhar O; Yakimenko, Zoya A; Svishcheva, Natalia V; Tagaev, Andrey A; Skladnev, Dmitry A; Arseniev, Alexander S

    2003-01-01

    Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution. PMID:14658801

  1. HN-NCA heteronuclear TOCSY-NH experiment for {sup 1}H{sup N} and {sup 15}N sequential correlations in ({sup 13}C, {sup 15}N) labelled intrinsically disordered proteins

    Wiedemann, Christoph; Goradia, Nishit; Häfner, Sabine [Leibniz Institute for Age Research, Fritz Lipmann Institute, Research Group Biomolecular NMR Spectroscopy (Germany); Herbst, Christian [Ubon Ratchathani University, Department of Physics, Faculty of Science (Thailand); Görlach, Matthias; Ohlenschläger, Oliver; Ramachandran, Ramadurai, E-mail: raman@fli-leibniz.de [Leibniz Institute for Age Research, Fritz Lipmann Institute, Research Group Biomolecular NMR Spectroscopy (Germany)

    2015-10-15

    A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue ‘i’ with that of residues ‘i−1’ and ‘i+1’ in ({sup 13}C, {sup 15}N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of {sup 1}J{sub CαN} and {sup 2}J{sub CαN} couplings to transfer the {sup 15}N{sub x} magnetisation from amino acid residue ‘i’ to adjacent residues via the application of a band-selective {sup 15}N–{sup 13}C{sup α} heteronuclear cross-polarisation sequence of ∼100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.

  2. Synthesis and NMR characterization of (15N)taurine [2-(15N)aminoethanesulfonic acid

    The title compound was prepared in three steps with 55% overall yield starting from potassium (15N)phthalimide. The synthetic route involved reaction with 1,2-dibromoethane, hydrolysis of the resulting N-(2-bromoethyl) (15N)phthalimide with HBr and treatment of the 2-bromoethyl(15N)amine thus formed with sodium sulphite. The product was characterized by 13C, 1H and 15N NMR spectroscopy. The absolute coupling constants of 15N with the 13C nuclei and the non-exchanging protons were determined and an unambiguous assignment of the proton signals obtained. (author)

  3. Hyperpolarized 15N-pyridine Derivatives as pH-Sensitive MRI Agents

    Weina Jiang; Lloyd Lumata; Wei Chen; Shanrong Zhang; Zoltan Kovacs; A. Dean Sherry; Chalermchai Khemtong

    2015-01-01

    Highly sensitive MR imaging agents that can accurately and rapidly monitor changes in pH would have diagnostic and prognostic value for many diseases. Here, we report an investigation of hyperpolarized 15N-pyridine derivatives as ultrasensitive pH-sensitive imaging probes. These molecules are easily polarized to high levels using standard dynamic nuclear polarization (DNP) techniques and their 15N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp 15N resona...

  4. Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks

    Kumar, Dinesh

    2013-12-01

    Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone 15N and 13C‧/13Cα chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone 15N(i) chemical shifts with that of 13C‧ (i - 1)/13Cα (i - 1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate 15N chemical shifts and (ii) reduces the signal overlap in F2(15N)-F3(1H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.

  5. The effect of organic matter and nitrification inhibitor on 15 N H4 and 15 N O3 absorption by the maize

    The effect of the forms 15 N H4 and 15 N O3 in presence or absence of organic matter and of the nitrification inhibitor AM (2-amino-4-chloro-6-methyl-pyrimidine) in dry matter weight and nitrogen content of the plant derived from soil and form fertilizer is studied. The experiment was carried out in greenhouse and the test plant was the hybrid Maize Centralmex . The fertilizers (15 N H4)2 S O4 and Na15 N O3, were added in two levels: 40 and 120 Kg N/ha, with 1,02% of N and 1,4% of 15 N in excess, respectively. Three soils of different physical and chemical characteristics were used; Regosol intergrade, Latosol Roxo and Podzolized de Lins e Marilia var. Marilia. (M.A.C.)

  6. Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods

    While extracting dynamics parameters from backbone 15N relaxation measurements in proteins has become routine over the past two decades, it is increasingly recognized that accurate quantitative analysis can remain limited by the potential presence of systematic errors associated with the measurement of 15N R1 and R2 or R1ρ relaxation rates as well as heteronuclear 15N-{1H} NOE values. We show that systematic errors in such measurements can be far larger than the statistical error derived from either the observed signal-to-noise ratio, or from the reproducibility of the measurement. Unless special precautions are taken, the problem of systematic errors is shown to be particularly acute in perdeuterated systems, and even more so when TROSY instead of HSQC elements are used to read out the 15N magnetization through the NMR-sensitive 1H nucleus. A discussion of the most common sources of systematic errors is presented, as well as TROSY-based pulse schemes that appear free of systematic errors to the level of 2′/R1′ ratios fit an axially symmetric diffusion tensor with a Pearson’s correlation coefficient of 0.97, comparable to fits obtained for backbone amide RDCs to the Saupe matrix.

  7. Fields of application and results of analytic procedures with 15N in pediatric alimentary research

    Investigation of protein metabolism in nutritional pediatric research by means of 15N tracer techniques has been relatively seldom used up to now. 15N labelled compounds for these purposes are not injurious to health. The technique is based on oral or intravenous application of the tracer substances and on 15N analysis of the urine fractions. The subsequent calculation of protein synthesis and breakdown rate, turnover and reutilisation of amino acids from protein breakdown as well as the size of the metabolic pool offers detailed information of protein metabolism. Determination of these parameters was performed in infants on mother's milk and formula feeding and on chemically defined diet. As an example of utilisation of D-amino acids for protein synthesis the 15N-D-phenylalanin retention on parenteral nutrition was found to be 33% of the applied dosis at an average. An oral 15N glycine loading test proved to be of value for the prediction of the therapeutic effect of human growth hormon in numerous types of dwarfism. Further application of 15N tracer technique dealt with utilisation of 15N urea for bacterial protein synthesis of the intestinal flora and with incorporation of 15N from 15N glycine and 15N lysine into the jejunal mucosa for measuring the enterocyte regeneration. (author)

  8. CPMG sequences with enhanced sensitivity to chemical exchange

    Improved relaxation-compensated Carr-Purcell-Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone 15N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc.121, 2331-2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from 1H-1H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional 15N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor

  9. Efficient identification of amino acid types for fast protein backbone assignments

    We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective 15N-labeling. The usefulness of selective incorporation of 15N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with 15N-labeled amino acids. Here we demonstrate that purification of the selectively 15N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins

  10. 15N in biological nitrogen fixation studies

    A bibliography with 298 references on the use of the stable nitrogen isotope 15N in the research on the biological fixation of dinitrogen is presented. The literature pertaining to this bibliography covers the period from 1975 to the middle of 1985. (author)

  11. Getting started with Backbone Marionette

    Armendariz, Raymundo

    2014-01-01

    This book is written with an easy-to-understand approach with the intention of giving small but concrete examples that will help you to quickly understand each component of Marionette. Follow along as we work together to build a practical application using Backbone Marionette.If you are a web application developer interested in using Backbone Marionette for a real-life project, then this book is for you. As a prerequisite, knowledge of JavaScript and a working knowledge of Backbone.js is required.

  12. 15N2 incorporation by rhizosphere soil

    Heterotrophic nitrogen fixation by rhizosphere soil samples from 20 rice cultivars grown under uniform field conditions was estimated employing 15N-tracer technique. Rhizosphere soil samples from different rice cultivars showed striking differences with regard to their ability to incorporate 15N2. Rhizosphere samples from rice straw-amended (3 and 6 tons/ha) soil exhibited more pronounced nitrogen-fixing activity than the samples form unamended soil; while the activity of the rhizosphere samples from soil receiving combined nitrogen (40 and 80 kg N/ha) was relatively low. However, the inhibitory effect of combined nitrogen was not expressed in the presence of rice straw at 6 tons/ha. Results suggest that plant variety, application of combined nitrogen and organic matter influence the rhizosphere nitrogen fixation. (orig.)

  13. Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

    Mai, W.; Hu, W; Wang, C; Cross, T A

    1993-01-01

    Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these con...

  14. Sources and transformations of N in reclaimed coastal tidelands: evidence from soil δ15N data

    Kwak, Jin-Hyeob; Choi, Woo-Jung; Lim, Sang-Sun; Lee, Seung-Heon; Lee, Sang-Mo; Chang, Scott X.; Jung, Jae-Woon; Yoon, Kwang-Sik; Choi, Soo-Myung

    2008-01-01

    Electrical conductivity of saturated soil extracts (ECe) in three reclaimed tideland (RTL) soils on the west coast of Korea decreased with time since reclamation, indicating natural desalinization through leaching of salts by precipitation water. Soil N concentration increased with decreasing ECe. With the increase in soil N concentration, the δ15N decreased, likely caused by the input of 15N-depleted N sources. As N2-fixing plant species were found in the oldest RTL, atmospheric N2 fixation likely contributed to the increase in soil N concentration in the oldest RTL. Negative δ15N (-7.1 to -2.0‰) of total inorganic N (NH4 ++NO3 -) and published data on N deposition near the study area indicate that atmospheric N deposition might be another source of N in the RTLs. Meanwhile, the consistently negative δ15N of soil NO3 - excluded N input from chemical fertilizer through groundwater flow as a potential N source, since NO3 - in groundwater generally have a positive δ15N. The patterns of δ15N of NH4 + (+2.3 to +5.1‰) and NO3 - (-9.2 to -5.0‰) suggested that nitrification was an active process that caused 15N enrichment in NH4 + but denitrification was probably minimal which would otherwise have caused 15N enrichment in NO3 -. A quantitative approach on N budget would provide a better understanding of soil N dynamics in the studied RTLs.

  15. 15N-labed glycine synthesis

    Claudinéia R. O. Tavares; José A. Bendassolli; Fernando Coelho; Carlos R. Sant Ana Filho; Clelber V. Prestes

    2006-01-01

    This work describes a method for 15N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of alpha-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia (15NH3). Special care was taken to avoid possible 15NH3 losses, since its production cost is high. In that respect, although the purchase cost of the 13N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an i...

  16. Fuzzy logic control of 15 N separation plant

    The process of 15 N separation by chemical exchange in Nitrox system is automatically maintained in the optimal operation conditions using a computerized control. The automatic control leads to a maximum production of 15 N with a minimum of raw materials and energy consumption.. The control objective was achieved by considering two forms of knowledge: 1. objective knowledge, which uses the control engineering based on mathematical model of the separation process; 2. subjective knowledge, which represents linguistic information, very difficult to quantify using classical mathematics - e.g., the rule of HNO3 solution and SO2 flow rates adjustment in order to maintain a proper height and position of chemical reaction zone in the product refluxer. The above mentioned two types of knowledge were coordinated in a logical way using fuzzy logic control system which has the possibility to handle simultaneously numerical data and linguistic knowledge. In order to map input data vector into a scalar output, i.e., numbers to numbers a front-end 'fuzzifier' and a rear-end 'defuzzifier' was added to the usual fuzzy logic model. The inference engine of the control system maps the input fuzzy set into the output one. The inferential procedure maintains the isotope separation process in the optimal operation conditions. (author)

  17. Effect of protein restriction on (15)N transfer from dietary [(15)N]alanine and [(15)N]Spirulina platensis into urea.

    Hamadeh, M J; Hoffer, L J

    2001-08-01

    Six normal men consumed a mixed test meal while adapted to high (1.5 g. kg(-1) x day(-1)) and low (0.3 g. kg(-1) x day(-1)) protein intakes. They completed this protocol twice: when the test meals included 3 mg/kg of [(15)N]alanine ([(15)N]Ala) and when they included 30 mg/kg of intrinsically labeled [(15)N]Spirulina platensis ([(15)N]SPI). Six subjects with insulin-dependent diabetes mellitus (IDDM) receiving conventional insulin therapy consumed the test meal with added [(15)N]Ala while adapted to their customary high-protein diet. Protein restriction increased serum alanine, glycine, glutamine, and methionine concentrations and reduced those of leucine. Whether the previous diet was high or low in protein, there was a similar increase in serum alanine, methionine, and branched-chain amino acid concentrations after the test meal and a similar pattern of (15)N enrichment in serum amino acids for a given tracer. When [(15)N]Ala was included in the test meal, (15)N appeared rapidly in serum alanine and glutamine, to a minor degree in leucine and isoleucine, and not at all in other circulating amino acids. With [(15)N]SPI, there was a slow appearance of the label in all serum amino acids analyzed. Despite the different serum amino acid labeling, protein restriction reduced the postmeal transfer of dietary (15)N in [(15)N]Ala or [(15)N]SPI into [(15)N]urea by similar amounts (38 and 43%, respectively, not significant). The response of the subjects with IDDM was similar to that of the normal subjects. Information about adaptive reductions in dietary amino acid catabolism obtained by adding [(15)N]Ala to a test meal appears to be equivalent to that obtained using an intrinsically labeled protein tracer. PMID:11440912

  18. Nitrogen cycling in an extreme hyperarid environment inferred from δ15N analyses of plants, soils and herbivore diet

    Díaz, Francisca P.; Matías Frugone; Gutiérrez, Rodrigo A.; Claudio Latorre

    2016-01-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ15N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ15N and δ13C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ15N va...

  19. Change of 15N natural abundance (δ15N) in a forest soil receiving elevated N deposition

    Natural abundance of 15N15N) has been used to interpret N mineralization in forest ecosystems. Forest litter typically has depleted δ15N values ranging from -8 to 0 per mille and δ15N values of organic N in forest soil profiles become more enriched with depth. This study investigated (1) the change of δ15N and total N with depth, and (2) the relation between the change of δ15N within the 0 to 10, 10 to 20 and 20 to 30 cm intervals of the mineral layer and the N mineralization rates in these layers

  20. The 15N-enrichment in dark clouds and Solar System objects

    Hily-Blant, Pierre; Faure, Alexandre; Quirico, Eric

    2013-01-01

    The line intensities of the fundamental rotational transitions of H13CN and HC15N were observed towards two prestellar cores, L183 and L1544, and lead to molecular isotopic ratios 140 6 14N/15N 6 250 and 140 6 14N/15N 6 360, respectively. The range of values reflect genuine spatial variations within the cores. A comprehensive analysis of the available measurements of the nitrogen isotopic ratio in prestellar cores show that molecules carrying the nitrile functional group appear to be systematically 15N-enriched com- pared to those carrying the amine functional group. A chemical origin for the differential 15N-enhance- ment between nitrile- and amine-bearing interstellar molecules is proposed. This sheds new light on several observations of Solar System objects: (i) the similar N isotopic fractionation in Jupiter's NH3 and solar wind N+; (ii) the 15N-enrichments in cometary HCN and CN (that might represent a direct inter- stellar inheritance); and (iii) 15N-enrichments observed in organics in primitive cosmoma...

  1. Application of unsymmetrical indirect covariance NMR methods to the computation of the (13)C (15)N HSQC-IMPEACH and (13)C (15)N HMBC-IMPEACH correlation spectra.

    Martin, Gary E; Hilton, Bruce D; Irish, Patrick A; Blinov, Kirill A; Williams, Antony J

    2007-10-01

    Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method. PMID:17729230

  2. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons

    Live, D.H.; Cowburn, D.

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence for hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neutrophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.

  3. Nitrogen cycling in an extreme hyperarid environment inferred from δ15N analyses of plants, soils and herbivore diet

    Díaz, Francisca P.; Frugone, Matías; Gutiérrez, Rodrigo A.; Latorre, Claudio

    2016-03-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ15N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ15N and δ13C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ15N values span the entire gradient, soil δ15N values show a positive correlation with aridity as expected. In contrast, foliar δ15N values and herbivore feces show a hump-shaped relationship with elevation, suggesting that plants are using a different N source, possibly of biotic origin. Thus at the extreme limits of plant life, biotic interactions may be just as important as abiotic processes, such as climate in explaining ecosystem δ15N values.

  4. Nitrogen cycling in an extreme hyperarid environment inferred from δ(15)N analyses of plants, soils and herbivore diet.

    Díaz, Francisca P; Frugone, Matías; Gutiérrez, Rodrigo A; Latorre, Claudio

    2016-01-01

    Climate controls on the nitrogen cycle are suggested by the negative correlation between precipitation and δ(15)N values across different ecosystems. For arid ecosystems this is unclear, as water limitation among other factors can confound this relationship. We measured herbivore feces, foliar and soil δ(15)N and δ(13)C values and chemically characterized soils (pH and elemental composition) along an elevational/climatic gradient in the Atacama Desert, northern Chile. Although very positive δ(15)N values span the entire gradient, soil δ(15)N values show a positive correlation with aridity as expected. In contrast, foliar δ(15)N values and herbivore feces show a hump-shaped relationship with elevation, suggesting that plants are using a different N source, possibly of biotic origin. Thus at the extreme limits of plant life, biotic interactions may be just as important as abiotic processes, such as climate in explaining ecosystem δ(15)N values. PMID:26956399

  5. 15N fractionation in star-forming regions and Solar System objects

    Wirström, Eva; Milam, Stefanie; Adande, Gilles; Charnley, Steven B.; Cordiner, Martin A.

    2015-08-01

    A central issue for understanding the formation and evolution of matter in the early Solar System is the relationship between the chemical composition of star-forming interstellar clouds and that of primitive Solar System materials. The pristine molecular content of comets, interplanetary dust particles and carbonaceous chondrites show significant bulk nitrogen isotopic fractionation relative to the solar value, 14N/15N ~ 440. In addition, high spatial resolution measurements in primitive materials locally show even more extreme enhancements of 14N/15N natal molecular cloud core and the outer protosolar nebula. Indeed, early chemical models of gas-phase ion-molecule nitrogen fractionation showed that HCN and HNC (nitriles) can hold significant 15N enrichments in cold dark clouds where CO is depleted onto dust grains. In addition, 15N fractionation in nitriles and amines (NH2, NH3) follow different chemical pathways. More recently we have shown that once the spin-state dependence in rates of reactions with H2 is included in the models, amines can either be enhanced or depleted in 15N, depending on the core’s evolutionary stage. Observed 15N fractionation in amines and nitriles therefore cannot be expected to be the same, instead their ratio is a potential chemical clock.Observations of molecular isotope ratios in dark cores are challenging. Limited published results in general show higher 15N/14N ratios in HCN and HNC than ammonia, but more measurements are necessary to confirm these trends. We will present recent results from our ongoing observing campaign of 14N/15N isotopic ratios in HCN, HNC and NH3 in dense cores and protostars which seem consistent with significant fractionation in nitriles as compared to other molecules in each object. The few 14N/15N ratios observed in N2H+ are similar to those in NH3, contrary to our model results which predict a significant 15N enhancement in N2 and N2H+. Model upgrades which may address this discrepancy will be

  6. Simultaneous acquisition of 13Cα–15N and 1H–15N–15N sequential correlations in proteins: application of dual receivers in 3D HNN

    We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable 13Cα transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D 13Cα–15N sequential correlations along with the standard 3D 15N–15N–1H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.

  7. 15N NMR spectroscopy of Pseudomonas cytochrome c-551

    15N-1H correlation spectroscopy with detection at the 1H frequency has been used at natural abundance to detect nitrogen nuclei bonded to protons in the ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429). Side-chain aromatic nitrogen, main-chain amides, and side-chain amides have been assigned to specific residues by comparison to previous proton assignments. Assignment ambiguities arising from overlap in the proton dimension have been resolved by examining spectra as a function of temperature and pH. Nitrogen chemical shifts are reported at pH 4.6 and 9.4 and three temperatures, 32, 50, and 60 degree C. Significant differences arise from the observed protein shifts and expected shifts in the random coil polypeptide

  8. Study of protein metabolism and cell proliferation using 15N

    Investigations of nitrogen and protein metabolism with the stable isotope 15N were carried out in 11 patients with arteriosclerosis and 7 healthy controls. After oral application of 3 g 15NH4Cl (95 At% 15N) per 70 kg body weight the incorporation of the isotope 15N in plasma proteins and blood cells and the 15N elimination in urine were followed up. Retardations of 15N elimination, an accelerated incorporation of 15N in fibrin and a retarded 15N incorporation in platelet protein were observed in patients with arteriosclerosis. The described method enables complex assertions about protein metabolism of the whole body and so represents a possibility to evaluate objectively the influence of an intervention on metabolism. (author)

  9. Backbone dynamics of the human CC-chemokine eotaxin

    Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. 15N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. 15N longitudinal (R1) and transverse (R2) auto relaxation rates, heteronuclear 1H-15N steady-state NOEs, and transverse cross-relaxation rates (ηxy) were obtained at 30 deg. C for all resolved backbone secondary amide groups using 1 H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (τm) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (Dparallel/Dperpendicular) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two β-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization

  10. 15N-{1H} NOE experiment at high magnetic field strengths

    The heteronuclear 15N-{1H} NOE values are typically determined by taking the ratio of 15N signal intensities recorded in the presence and absence of 1H saturation prior to evolution of 15N magnetization. Since the intensity ratio of two independent experiments is taken, complete recovery of 15N magnetization during the scan repetition delay is critical to obtain reliable NOE values. Because it may not be practical to wait for the complete recovery of magnetization at high magnetic fields, Solomon equations may be used to correct measured NOE values. Here, based on experiments and simulations, we show that since the cross-correlation between 1H-15N dipole and 15N chemical shift anisotropy becomes significant at high fields for small or deuterated proteins, measured NOE values can not be accurately corrected based on the Solomon equations. We also discuss ranges of rotational correlation times and proton spin-flip rate, in which the NOE values can be corrected by the equations

  11. 1H, 13C, and 15N backbone and side chain resonance assignments of thermophilic Geobacillus kaustophilus cyclophilin-A

    Holliday, Michael; Zhang, Fengli; Isern, Nancy G.; Armstrong, Geoffrey S.; Eisenmesser, Elan Z.

    2014-04-01

    Cyclophilins catalyze the reversible peptidyl-prolyl isomerization of their substrates and are present across all kingdoms of life from humans to bacteria. Although numerous biological roles have now been discovered for cyclophilins, their function was initially ascribed to their chaperone-like activity in protein folding where they catalyze the often rate-limiting step of proline isomerization. This chaperone-like activity may be especially important under extreme conditions where cyclophilins are often over expressed, such as in tumors for human cyclophilins {Lee, 2010 #1167}, but also in organisms that thrive under extreme conditions, such as theromophilic bacteria. Moreover, the reversible nature of the peptidyl-prolyl isomerization reaction catalyzed by cyclophilins has allowed these enzymes to serve as model systems for probing the role of conformational changes during catalytic turnover {Eisenmesser, 2002 #20;Eisenmesser, 2005 #203}. Thus, we present here the resonance assignments of a thermophilic cyclophilin from Geobacillus kaustophilus derived from deep-sea sediment {Takami, 2004 #1384}. This thermophilic cyclophilin may now be studied at a variety of temperatures to provide insight into the comparative structure, dynamics, and catalytic mechanism of cyclophilins.

  12. MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data

    MERA (Maximum Entropy Ramachandran map Analysis from NMR data) is a new webserver that generates residue-by-residue Ramachandran map distributions for disordered proteins or disordered regions in proteins on the basis of experimental NMR parameters. As input data, the program currently utilizes up to 12 different parameters. These include three different types of short-range NOEs, three types of backbone chemical shifts (15N, 13Cα, and 13C′), six types of J couplings (3JHNHα, 3JC′C′, 3JC′Hα, 1JHαCα, 2JCαN and 1JCαN), as well as the 15N-relaxation derived J(0) spectral density. The Ramachandran map distributions are reported in terms of populations of their 15° × 15° voxels, and an adjustable maximum entropy weight factor is available to ensure that the obtained distributions will not deviate more from a newly derived coil library distribution than required to account for the experimental data. MERA output includes the agreement between each input parameter and its distribution-derived value. As an application, we demonstrate performance of the program for several residues in the intrinsically disordered protein α-synuclein, as well as for several static and dynamic residues in the folded protein GB3

  13. MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data

    Mantsyzov, Alexey B. [M.V. Lomonosov Moscow State University, Faculty of Fundamental Medicine (Russian Federation); Shen, Yang; Lee, Jung Ho [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Hummer, Gerhard [Max Planck Institute of Biophysics (Germany); Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2015-09-15

    MERA (Maximum Entropy Ramachandran map Analysis from NMR data) is a new webserver that generates residue-by-residue Ramachandran map distributions for disordered proteins or disordered regions in proteins on the basis of experimental NMR parameters. As input data, the program currently utilizes up to 12 different parameters. These include three different types of short-range NOEs, three types of backbone chemical shifts ({sup 15}N, {sup 13}C{sup α}, and {sup 13}C′), six types of J couplings ({sup 3}J{sub HNHα}, {sup 3}J{sub C′C′}, {sup 3}J{sub C′Hα}, {sup 1}J{sub HαCα}, {sup 2}J{sub CαN} and {sup 1}J{sub CαN}), as well as the {sup 15}N-relaxation derived J(0) spectral density. The Ramachandran map distributions are reported in terms of populations of their 15° × 15° voxels, and an adjustable maximum entropy weight factor is available to ensure that the obtained distributions will not deviate more from a newly derived coil library distribution than required to account for the experimental data. MERA output includes the agreement between each input parameter and its distribution-derived value. As an application, we demonstrate performance of the program for several residues in the intrinsically disordered protein α-synuclein, as well as for several static and dynamic residues in the folded protein GB3.

  14. Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.

    Okar, D A; Live, D H; Devany, M H; Lange, A J

    2000-08-15

    The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of

  15. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  16. Synthesis of [α-15N]-dl-tryptophan

    [α-15N]-dl-tryptophan was synthesized by the use of Al-Ni alloy catalytic hydrogenation from 15N-glycine via several steps. The overall yield of the final product was 46.9% and the abundance of 15N was about 93%. The physicochemical properties of the synthetic compound obtained were the same as those of the standard tryptophan. Its structure were confirmed by the elemental analyses, MS, UV and paper chromatography

  17. 15N-ammonium test in clinical research

    By use of the 15N-ammonium test the liver function is investigated under influence of hormonal contraceptives in women and in liver diseases in children. With the described noninvasive nonradioactive isotope test the ammonia detoxification capability and the urea synthesis capacity of the liver is determined by measuring of the 15N excretion in ammonia and urea in urine after oral administering of 15N-ammonium chloride. The 15N-ammonium test shows a significant influence of the hormonal contraceptives on the liver function and gives diagnostic evidence for liver diseases in children. (author)

  18. 15N tracer methodology for absorption studies in nutrition research

    Proceeding from 15N analyses, 15N tracer methods, and a model of protein metabolism it is shown that the nitrogen balance is a useful concept for expressing the relationship between the overall nitrogen intake of the body and the nitrogen excretion. After admistering low doses of 15N-labelled substances like protein and amino acids, the kinetics of digestion and absorption can be followed by measuring the 15N abundance in serum and urine of patients. A significant delay in the nitrogen absorption indicates gastrointestinal disorders

  19. Studies with 15N-lysine in colostomized hens. 1

    0.2% L-lysine with an atom-% 15N excess (15N') of 48% were given per day through a throat probe to three colostomized laying hybrids in addition to a pelleted ration of 120 g per animal and day. In the following 4 days unlabelled L-lysine was given. As the labelled lysine was given three times a day, the development of 15N' excretion could be pursued. 80 minutes after the 15N'-lysine dose a distinct atom-% 15N' could be detected in urine. 6 hours after the 15N' application 2.9%, 4.2% and 2.7%, resp. of the applied 15N' amount in urine were found. 8 days after the beginning of the experiment the excretion of 15N' in urine was 17.5% on the average of the consumed 15N' amount. 44% of the nitrogen in the ration, however, was excreted in urine. The results show that the lysine N is excreted to a considerably lower extent in urine than the nitrogen in the remaining ration. (author)

  20. 15N balance in wheat-moong-soybean cropping sequence

    Field experiments were conducted to study the effect of FYM and S on fertilizer 15N balance in wheat-moong-soybean cropping sequence, with the main emphasis on partial substitution of chemical fertilizer N through FYM. Response to partial substitution of N was observed in the first crop of the sequence. FYM substitution at higher level (50%) resulted in reduction of wheat yield, but 25% substitution of recommended N through FYM increased wheat yield. Total fertilizer N recovery by three crops wheat, moong and soybean grown in sequence ranged between 39 to 55 per cent of which 35 to 41 per cent was utilized by the first crop and 4 to 14 per cent by the second and third crops together while 21 to 36 per cent of the fertilizer N applied to wheat was present in soil after growing three crops. Fertilizer N recovery in soil plant system was 61 to 91 per cent. Higher fertilizer N recovery was associated with higher rate of substitution of FYM for chemical fertilizer. FYM boosted fertilizer N recovery and higher soil retentivity. Sulphur application had no significant effect on per cent residual fertilizer N retention in soil. (author)

  1. Studies with 15N-lysine in colostomized hens. 6

    3 colostomized laying hybrides received 91.40 mg L-lysine-15N-excess (15N') each over a period of 4 days in a metabolism experiment with 15N-lysine. After another 4 days, during which the hens received the same rations supplemented by commercial L-lysine, the animals were butchered and divided into individual fractions. After hydrochloric hydrolysis of organs and tissues the heavy nitrogen of lysine, histidine and arginine were separated, quantitatively evaluated, processed and measured with an emission spectrometer. Atom-% 15N' on an average amounted to 0.20 in the liver, 0.16 in the kidneys, 0.06 in the flesh and 0.05 in the bones. Of the rediscovered 15N' applied, feces contained 8.1 %, urine 18.3 %, the eggs 24.3 %, the blood 4.9 %, the flesh 20.5 %, the bones 5.2 %, the gastrointestinal tract with its contents 4.5 %, the liver 3.5 %, the kidneys 0.9 %, the reproductive organs 3.7 %, and the rest 6.1 %. The quota of rediscovery of the 15N' applied was 95.7 %. 62 % of the total 15N' was rediscovered in eggs, body and feces as lysine 15N'. There was significantly more 15N' in all arginine fractions than in histidine. The quota of the lysine-15N' of the total 15N' differed considerably in the fractions: < 40 % bones and blood; 48-56 % gastrointestinal tract, feces, oviduct, kidneys; 62-63 % remaining ovary, rest; 69-71 % eggs, flesh, liver. It could be proved that the α-amino group of lysine is to a large extent incorporated into other amino acids. Further proof that the amino acid metabolism proceeds in two phases was submitted, i.e. higher amounts of amino acids previously deposited in the body are used for egg synthesis. (author)

  2. Comparison of {sup 15}N- and {sup 13}C-determined parameters of mobility in melittin

    Zhu Lingyang [University Indianapolis, Department of Physics, Indiana University Purdue (United States); Prendergast, Franklyn G. [Mayo Foundation, Department of Pharmacology (United States); Kemple, Marvin D. [University Indianapolis, Department of Physics, Indiana University Purdue (United States)

    1998-07-15

    Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by {sup 15}N and {sup 13}C NMR. Specifically, inverse-detected {sup 15}N T{sub 1} and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 deg. C enriched with {sup 15}N at six amide positions and in the Trp{sup 19} side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and {alpha}-helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: {tau}{sub m}, the correlation time for the overall rotation; S{sup 2}, a site-specific order parameter which is a measure of the amplitude of the internal motion; and {tau}{sub e}, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the {sup 15}N measurements and from {sup 13}C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678-1688]. {tau}{sub m} and {tau}{sub e} values were consistent from data on the two nuclei. In the MLT monomer, S{sup 2} values for the backbone N-H and C{alpha}-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C{alpha}-H order parameters. The Trp side-chain N-H and C-H order parameters, and {tau}{sub e} values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.

  3. Highly automated protein backbone resonance assignment within a few hours: the strategy and software package

    Sequential resonance assignment represents an essential step towards the investigation of protein structure, dynamics, and interaction surfaces. Although the experimental sensitivity has significantly increased in recent years, with the availability of high field magnets and cryogenically cooled probes, resonance assignment, even of small globular proteins, still generally requires several days of data collection and analysis using standard protocols. Here we introduce the BATCH strategy for fast and highly automated backbone resonance assignment of 13C, 15N-labelled proteins. BATCH makes use of the fast data acquisition and analysis tools BEST, ASCOM, COBRA, and HADAMAC, recently developed in our laboratory. An improved Hadamard encoding scheme, presented here, further increases the performance of the HADAMAC experiment. A new software platform, interfaced to the NMRView software package, has been developed that enables highly automated NMR data processing and analysis, sequential resonance assignment, and 13C chemical shift extraction. We demonstrate for four small globular proteins that sequential resonance assignment can be routinely obtained within a few hours, or less, in a highly automated and robust way

  4. Multinuclear NMR of 15 N labelled organic molecules

    The paper presents the application of multinuclear NMR techniques to the study of 15 N labeled organic molecules. There are some important points of great interest in such type of research, namely, structure determination, i.e. location of the 15 N in molecule and determination of 15 N concentration in order to obtain quantitative results about the intramolecular short and long range interaction. Different NMR techniques were used in the study of 13 C, 1 H and 15 N. Obtaining the 15 N NMR signal imposes some special preparation of the spectrometer. First, we had to manage a very large spectral window (-400 to +1200 ppm) which makes difficult finding the signal. Secondly, in the condition of proton decoupling, in a very large band, a decrease of the signal can occur due to the NOE negative effect. To avoid this effect, other decoupling method, called 'inverse gated 1 H decoupling' was used. As a reference, for 15 N, we used CH3NO2, fixed at 0 ppm. In order to find the suitable spectral window we used the formamide (15 N). The results of obtaining the 15 N-labeled procaine are presented. (author)

  5. 15N Fractionation in Star-Forming Regions and Solar System Objects

    Wirstrom, Eva; Milam, Stefanie; Adande, GIlles; Charnley, Steven; Cordiner, Martin

    2015-01-01

    A central issue for understanding the formation and evolution of matter in the early Solar System is the relationship between the chemical composition of star-forming interstellar clouds and that of primitive Solar System materials. The pristinemolecular content of comets, interplanetary dust particles and carbonaceous chondrites show significant bulk nitrogen isotopic fractionation relative to the solar value, 14N15N 440. In addition, high spatial resolution measurements in primitive materials locally show even more extreme enhancements of 14N15N 100.

  6. 15N2 incorporation and acetylene reduction by azospirillum isolated from rice roots and soils

    Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by 15N2 incorporation and C2H2 reduction. C2H2 reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in 15N2 incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N2-fixing ability and that plant genotype is of importance for optimal associations. (orig.)

  7. 1H, 15N and 13C NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450

    The FMN-binding domain of human NADPH-cytochrome P450 reductase,corresponding to exons 3-;7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15Nand 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six α-helices and fiveβ-strands. The global fold was deduced from the long-range NOE sun ambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating α/β type, with the fiveβ-strands arranged into a parallel β-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchordomain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-bindingdomain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site

  8. A direct electrifying algorithm for backbone identification

    This paper proposes a new algorithm for identifying backbones in the application of percolation theory. This algorithm is based on the current-carrying definition of backbone and is carried out on the predetermined spanning cluster. It is fairly easy to implement and further parallelize. The efficiency is enhanced by the fact that the conductivity of a percolating system can be obtained in the same processing of backbone identification. The critical exponents of backbone mass, red bonds (sites) and conductivity obtained by this algorithm are in very good agreement with the existing results

  9. 3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

    Krejcirikova, Anna; Tugarinov, Vitali, E-mail: vitali@umd.edu [University of Maryland, Department of Chemistry and Biochemistry (United States)

    2012-10-15

    The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-{sup 2}H,{sup 15}N,{sup 13}C; Ile{delta}1-{l_brace}{sup 13}CH{sub 3}{r_brace}; Leu,Val-{l_brace}{sup 13}CH{sub 3}/{sup 12}CD{sub 3}{r_brace}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-{Delta}YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40 Degree-Sign C to improve the sensitivity of the measurements and alleviate the overlap of {sup 1}H-{sup 15}N correlations in the abundant {alpha}-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of {sup 1}H-{sup 15}N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D {sup 1}H-{sup 15}N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected 'out-and-back' HMCM(CG)CBCA experiment, and (3) strong sequential HN-HN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578-195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.

  10. Application of 15N in biochemistry, agriculture and medicine

    The compendium on application of 15N in the biosciences comprises 7 chapters. The 1st chapter comprehends introductory remarks on isotopes in general and on nitrogen isotopes in particular. In the 2nd chapter fundamentals of 15N tracer techniques are discussed. The 3rd chapter deals with experiment programs and the evaluation of experiments. The methodology of sample preparation as well as of isotope analysis is treated in chapter 4. The chapters 5 to 7 deal with the application of 15N as tracer in biochemistry, agricultural research and medicine, resp. Relevant literature is added to each chapter

  11. Struktur- und Bindungsuntersuchungen nichtextrahierbarer 15 N- und 14 C-Simazinrückstände im Boden

    Berns, Anne Elisabeth

    2003-01-01

    The aim of the presented study was the characterization of the structure and binding modes of non-extractable residues (NER) of the triazine herbicide simazine. The chemical environments of unaltered as well as metabolized simazine compounds can be observed directly in soil or compost matrix by 15N-NMR spectroscopy. As the 15N-isotope has a very low sensitivity and natural abundance 15N-labeled simazine was used. To further enhance the signal to noise ratio and sensitivity of the NMR experime...

  12. 1H, 13C, and 15N resonance assignment of the ubiquitin-like domain from Dsk2p

    Chen, Tony; Zhang, Daoning; Matiuhin, Yulia; Glickman, Michael; Fushman, David

    2008-01-01

    The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for 1H, 15N, and 13C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2’s interactions with proteasomal receptors and its role as ...

  13. Reduced Dimensionality tailored HN(C)N Pulse Sequences for Efficient Backbone Resonance Assignment of Proteins through Rapid Identification of Sequential HSQC peaks

    Kumar, Dinesh

    2013-01-01

    Two novel reduced dimensionality (RD) experiments -(4,3)D-hNCOcaNH and (4,3)D-hNcoCANH- have been presented here to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. The experiments basically represent an improvisation of previously reported HN(C)N experiment [Panchal et. al., J. Biomol. NMR. (2002), 20 (2), 135-147] and exploit the simple reduced dimensionality NMR concept [Szyperski et. al. (2002), Proc. Natl. Acad. Sci. U.S.A. 99(12), 8009-8014] to achieve (a) higher dispersion and resolution along the co-evolved F1 dimension and (b) rapid identification of sequential HSQC peaks on its F2(15N)- F3(1H) planes. The current implementation is based on the fact that the linear combination of 15N and 13CO/13Ca chemical shifts offers relatively better dispersion and randomness compared to the individual chemical shifts; thus enables the assignment of crowded HSQC spectra by resolving the ambiguities generally encountered in HNCN based assignment protocol because of ...

  14. Stereospecific assignments of glycine in proteins by stereospecific deuteration and {sup 15}N labeling

    Hansen, A.P.; Curley, R.W. Jr.; Panigot, M.J.; Fesik, S.W. [Ohio State Univ., Columbus, OH (United States)

    1994-12-01

    Stereospecific assignments are important for accurately determining the three-dimensional structures of proteins through the use of multidimensional NMR techniques. It is especially important to stereospecifically assign the glycine {alpha}-protons in proteins because of the potential for different backbone conformations of this residue. These stereospecific assignments are critical for interpreting the {sup 3}J{sub NH,{alpha}H} coupling constants and NOEs involving the glycine {alpha}-protons that determine the conformation of this part of the protein. However, it is often difficult to unambiguously obtain the stereospecific assignments for glycine residues by using only NOE data. In this poster, we present a method for unambiguous, stereospecific assignment of the {alpha}-protons of glycine residues. This method involves synthesis of stereo-specifically deuterated and {sup 15}N-labeled Gly using a slightly modified procedure originally described by Woodard and coworkers for the stereoselective deuteration of glycine. The stereospecifically deuterated and {sup 15}N-labeled Gy has been incorporated into recombinant proteins expressed in both bacterial systems (FKBP) and mammalian cells (u-PA). Two- and three-dimensional isotope-filtered and isotope-edited NMR experiments were used to obtain the stereospecific assignments of the glycine {alpha}-protons for these proteins.

  15. The absorption, utilization and distribution of nitrate 15N and ammonium 15N in Populus Tomentosa seedlings

    Effects of different nitrogen sources (NO3-, NH4+) on the absorption, distribution and utilization of nitrogen on Populus tenement's seedlings (clone 50) was studied by using the 15N trace technique. Results showed that the Populus tenement's seedlings had the same nitrogen take up pattern: tissue nitrogen content grew up after fertilization, remarkbaly rising up after one week and reached peak after 28 days. Although the treatments are different, the tissue N content was about the same between 0.6g · plant-1. The maximum absorption of NO3-15N and NH4-15N was 0.26g · plant-1 and 0.12g · plant -1, which accounted for 39.15% and 19.95% of total nitrogen, respectively. The nitrogen use efficiency (NUE) of two nitrogen sources varied gignificantly. The maximum NUE of NO3-15N reached 25.83%, nearly twice of that of NH4-15N (12.03%). Hence we conclude that Populus tomentosa seedlings (clone 50) prefer to absorb NO3-. Nitrogen distribution rate changed obviously among different organs and the trend was leaf>root>stem. In the leaf, the distribution of NO3-15N was higher than that of NH4-15N. (authors)

  16. Green Network Planning Model for Optical Backbones

    Gutierrez Lopez, Jose Manuel; Riaz, M. Tahir; Jensen, Michael;

    2010-01-01

    Communication networks are becoming more essential for our daily lives and critically important for industry and governments. The intense growth in the backbone traffic implies an increment of the power demands of the transmission systems. This power usage might have a significant negative effect...... an analytical model to consider environmental aspects in the planning stage of backbones design....

  17. Monitoring the refinement of crystal structures with 15N solid-state NMR shift tensor data

    The 15N chemical shift tensor is shown to be extremely sensitive to lattice structure and a powerful metric for monitoring density functional theory refinements of crystal structures. These refinements include lattice effects and are applied here to five crystal structures. All structures improve based on a better agreement between experimental and calculated 15N tensors, with an average improvement of 47.0 ppm. Structural improvement is further indicated by a decrease in forces on the atoms by 2–3 orders of magnitude and a greater similarity in atom positions to neutron diffraction structures. These refinements change bond lengths by more than the diffraction errors including adjustments to X–Y and X–H bonds (X, Y = C, N, and O) of 0.028 ± 0.002 Å and 0.144 ± 0.036 Å, respectively. The acquisition of 15N tensors at natural abundance is challenging and this limitation is overcome by improved 1H decoupling in the FIREMAT method. This decoupling dramatically narrows linewidths, improves signal-to-noise by up to 317%, and significantly improves the accuracy of measured tensors. A total of 39 tensors are measured with shifts distributed over a range of more than 400 ppm. Overall, experimental 15N tensors are at least 5 times more sensitive to crystal structure than 13C tensors due to nitrogen’s greater polarizability and larger range of chemical shifts

  18. Study on synthesis of 15N-hydrazine hydrate

    The 15N labeled hydrazine hydrate is a strong reducing agent in the synthesis procedure of stable isotope labeled compounds, and it has been widely used in the isotope-labeled pharmaceutical synthesis. The reaction conditions of 15N labeled hydrazine hydrate were mainly investigated by single-factor design, and the following optimized conditions were obtained: the concentration of available chlorine was 115-120 g/L, the chlorination re- action time was 30∼40 min, the reflux time was 7 min, and the mass ratio of material was m(catalyst) : m (urea) = 1.0 : 10.0, and the yield of 15N labeled hydrazine hydrate was 76.1%, the abundance of 15N was 99.20%. (authors)

  19. Protein-Backbone Thermodynamics across the Membrane Interface.

    Bereau, Tristan; Kremer, Kurt

    2016-07-01

    The thermodynamics of insertion of a protein in a membrane depends on the fine interplay between backbone and side-chain contributions interacting with the lipid environment. Using computer simulations, we probe how different descriptions of the backbone glycyl unit affect the thermodynamics of insertion of individual residues, dipeptides, and entire transmembrane helices. Due to the lack of reference data, we first introduce an efficient methodology to estimate atomistic potential of mean force (PMF) curves from a series of representative and uncorrelated coarse-grained (CG) snapshots. We find strong discrepancies between two CG models, Martini and PLUM, against reference atomistic PMFs and experiments. Atomistic simulations suggest a weak free energy of insertion between water and a POPC membrane for the glycyl unit, in overall agreement with experimental results despite severe assumptions in our calculations. We show that refining the backbone contribution in PLUM significantly improves the PMF of insertion of the WALP16 transmembrane peptide. An improper balance between the glycyl backbone and the attached side chain will lead to energetic artifacts, rationalizing Martini's overstabilization of WALP's adsorbed interfacial state. It illustrates difficulties associated with free-energy-based parametrizations of single-residue models, as the relevant free energy of partitioning used for force-field parametrization does not arise from the entire residue but rather the solvent-accessible chemical groups. PMID:27138459

  20. Studies with 15N-Lysine in colostomized hens. 4

    Each of 3 colostomized laying hens received per os 0.2% L-lysine with 48 atom-% 15N excess (15N') labelled in α-position in addition to a pelleted laying hen ration of 120 g over a period of 4 days. On the following 4 days they received equal amounts of unlabelled lysine. The eggs laid during the 8 days of the experiment were separated into the egg white, the yolk and the eggshell, and the total and heavy nitrogen in the individual fractions were determined. Above that, 17 amino acids and their atom-%15N' were determined in the 19 samples of the white and yolk of egg. Of the total 15N' from the lysine fed in the 4 days, 10.1% were found in the yolk, 10.5% in the egg white and 1.1% in the eggshells of the eggs laid during the 8 days of the experiment. 85% of the total amino acid 15N' of the yolk and 86% of the egg white detected to be lysine 15N'. The 15N' amount of the other 16 amino acids was mainly concentrated in the two acid and basic amino acids. Approximately 50% of the non-lysine 15N' in the egg are contained in aspartic acid, glutamic acid, histidine and arginine. A very low incorporation of the labelled lysine only could be detected in the aromatic and sulphur-containing amino acids from both the yolk and the egg white 43% of the 15N' was detected in the 10 essential and semi-essential (except lysine) and 57% in the 6 non-essential amino acids of the yolk and 52% and 48% resp. of the egg white. One can summarise that the incorporation of 15N' into the egg shows the same development as that of the labelled amino acids of the wheat protein and that 15% of the lysine 15N' could be detected in the 16 other amino acids. (author)

  1. Balance study of the fate of 15N fertilizer

    An interim report is presented on a series of experiments with wooden box-type lysimeters (60 cm x 60 cm x 70 cm) loaded with a sandy soil, a loess soil and straw-amended soil. The lysimeters support crops rotated over a five-year period to be studied - potato, barley, sugar-beet, barley (with winter rape) and finally (1979) potato. Each lysimeter received split applications of urea at total rates of 0, 50 or 100 kg.ha-1. The effects of soil residues of the herbicide monolinuron were also studied. The report deals with data collected during the first three years of the planned experiments (1975 - 1977 inclusive). 15N-labelled urea (47 atom 15N% excess) was initially used but in some experiments this was followed by applications of unlabelled urea in order to study the fate of the residual 15N in the subsequent years. The results to date indicated that in the first year highest recoveries in the plant of the applied 15N obtained on the sandy soil. The low recoveries of 15N in the subsequent years when unlabelled urea was supplied also indicated significant storage by soil or root organic matter of the applied 15N. Compared with the control (zero application of urea nitrogen), potato took up more total nitrogen in the presence of fertilizer including more of the unlabelled soil pool nitrogen. Analyses of the soil profiles in terms of total soil nitrogen and fertilizer-derived nitrogen (on the basis of 15N assays) indicated leaching of the labelled nitrogen down the soil profile in all cases during the three-year period. Analysis of NO3-N in leachates confirmed the presence of labelled urea-derived nitrogen. (author)

  2. Effects of compost application on fruits yields, sugar and mineral contents and δ15N values of tomato fruits

    We examined the effects of chemical fertilizer and compost application on the yields and sugar and mineral content of tomato (Lycopersicon esculentum Mill. Saturn) in an isolated bed. Five treatments were conducted within a two-year period of 4 continuous croppings. CDU and LSR (Low-sulfate slow-release fertilizer) were used for the chemical fertilizer plots. A mixture of cattle manure and CDU (CM + CDU), a mixture of poultry manure and CDU (PM + CDU), and mixture of cattle and poultry manure (PM + CM) plots were arranged as compost-using plots. We also measured the δ15N values of tomatoes and the soils of each treatment, and estimated the correlation of the δ15N values between fruits and soil to certify compost applied products. We did not find any reproductive differences in the yield or sugar content among the treatments. As to inorganic content of tomatoes, there were no significant differences except for Mg content among the plots. These results showed that it is difficult to assay regular benefit of organic fertilizer application to tomato yields and quality. On the other hand, δ15N values of tomato fruits showed significant differences among fertilizer applications. δ15N values of the chemical fertilizer were +1.6 per mille and -1.1% for CDU and LSR, respectively. Those of mixture of chemical and compost were +12.2 per mille and +11.2 per mille for CM + CDU and PM + CDU, respectively. The mixture of PM and CM showed the highest δ15N values (17.9 per mille) among the treatments. δ15N values of the soils and fruits reflected those of the fertilizers and were positively correlated (R2 = 0.89). It may be possible to use δ15N values as an indicator of organic products by setting the threshold point, e.g. +5.0 per mille, to distinguish them from the products cultivated with chemical fertilizer. (author)

  3. ~(15)N Isotope Used for Study of Groundwater Nitrogen Pollution in Shijiazhuang City, China

    2004-01-01

    Shijiazhuang City is the capital of Hebei province, China. Groundwater is the major water supply source for living and industry need of the city. Due to a rapid increase of population and development of industry and agriculture, a series of groundwater environmental problems are created. In the paper, the situation of groundwater pollution in Shijiazhuang city is reported. Based on the groundwater chemical data and ~(15)N measurement results both on groundwater and soils, the reason of groundwater nitra...

  4. Utilization of 15N-urea in laying hens. 3

    In 3 colostomized laying hens the incorporation of heavy nitrogen from urea into the amino acids of the 21 eggs laid during the 8-day experiment was determined. In these eggs the content of 15 amino acids was ascertained separately in white and yolk of the eggs and their atom-% 15N excess (15N') was determined. The heavy nitrogen could be detected in all amino acids investigated. The incorporation of 15N' into the essential amino acids of the white and yolk of eggs is very low. Of the 15N' amount of the urea applied 0.18% could be detected in the 9 essential amino acids of the white of egg and 0.12% in those of the yolk. For the 6 analyzed nonessential amino acids the rediscovery quota of 15N' in the white of egg was 0.50% and in the yolk 0.81% is that the NPN-source urea is insignificant for egg protein synthesis. (author)

  5. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M. [National Institutes of Health, Bethesda, MD (United States)

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  6. Exercise: The Backbone of Spine Treatment

    Full Text Available Exercise: The Backbone of Spine Treatment | View Video Back Purchase Video Struggling with Low Back Pain? Many people are surprised to learn that carefully selected exercise can ...

  7. Exercise: The Backbone of Spine Treatment

    Exercise: The Backbone of Spine Treatment | View Video Back Purchase Video Struggling with Low Back Pain? Many people are surprised to learn that carefully selected exercise can actually reduce back pain. Some exercises can ...

  8. Exercise: The Backbone of Spine Treatment

    Full Text Available Exercise: The Backbone of Spine Treatment | View Video Back Purchase Video Struggling with Low Back Pain? Many people are surprised to learn that carefully selected exercise can actually reduce back pain. Some exercises can ...

  9. Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c{sub 552} and its b-type variant

    Tozawa, Kaeko; Ferguson, Stuart J.; Redfield, Christina, E-mail: christina.redfield@bioch.ox.ac.uk [University of Oxford, Department of Biochemistry (United Kingdom); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [University of Oxford, Department of Chemistry (United Kingdom)

    2015-06-15

    Cytochrome c{sub 552} from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, {sup 15}N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high {sup 15}N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

  10. 15N-labelled pyrazines of triterpenic acids

    Triterpenoid pyrazines from our research group were found selectively cytotoxic on several cancer cell lines with IC50 in low micromolar range. This sparked our interest in preparing their labeled analogs for metabolic studies. In this work, we prepared a set of non-labeled pyrazines from seven triterpenoid skeletal types along with their 15N labelled analogs. In this work, we present the synthesis and characterization of the target 15N labelled pyrazines. Currently, these compounds are being studied in complex metabolic studies. (author)

  11. The 15N ground state studied with elastic electron scattering

    The C0 elastic electron scattering form factor of 15N has been measured over a momentum transfer range q = 0.4-3.2 fm-1. From these form factor data the ground state charge density and its RMS radius (2.612±0.009 fm) were determined. This charge density as well as its difference with that of 16O were compared to recent large-basis shell-model calculations. Although these calculations describe the individual charge density reasonably, the difference between 16O and 15N cannot be reproduced satisfactorily. (orig.)

  12. 15N magnetic resonance of aqueous imidazole and zinc(II)-imidazole complexes. Evidence for hexacoordination

    15N NMR chemical shifts of doubly labeled [15N)imidazole permit evaluation of hydrogen bonding, proton association, and Zn(II) complex formation in homogeneous solution. The 15N resonant frequency in aqueous solutions of imidazole at pH 9-12 is independent of imidazole concentration, suggesting insignificant self-association via hydrogen bonding involving the N3 lone pair and the N1 proton of a neighboring molecule. Protonation at N3 (pH less than 5) produces a 31.2-ppM diamagnetic shift and deprotonation at N1 (pH greater than 13) an approximately20-ppM paramagnetic shift relative to neutral aqueous imidazole. Those shifts are very large compared to the approximately +-0.5-ppM uncertainty in the 15N shift measurements. In solutions of Zn2+ and imidazole the 15N resonance in ZnIm/sub i/2+ complexes (Im = imidazole) is diamagnetically shifted by 10 to 20 ppM relative to neutral aqueous imidazole. Over a range of ratios of total imidazole to total zinc such that the average number of complexed imidazole molecules per Zn2+ (anti ν) is approximately 3.5, or less, the shift data are well interpreted by a four-species model (i = 1-4) using stepwise formation constants from the literature. Significant deviations from that model at anti ν greater than 3.5 require that higher species (e.g., ZnIm52+ and ZnIm62+) be considered. A six-species model with reasonable formation constants for the fifth and sixth complexes provides satisfactory interpretation of all data. Implications of those observations with respect to biologically active zinc(II) proteins are considered. 2 tables, 4 figures

  13. 15N Solid-State NMR as a Probe of Flavin H-bonding

    Cui, Dongtao; Koder, Ronald L.; Dutton, P. Leslie; Miller, Anne-Frances

    2011-01-01

    Flavins mediate a wide variety of different chemical reactions in biology. To learn how one cofactor can be made to execute different reactions in different enzymes, we are developing solid-state NMR (SSNMR) to probe the flavin electronic structure, via the 15N chemical shift tensor principal values (δii). We find that SSNMR has superior responsiveness to H-bonds, compared to solution NMR. H-bonding to a model of the flavodoxin active site produced an increase of 10 ppm in the δ11 of N5 altho...

  14. Detection of organic sulfur by [sup 15]N and [sup 19]F NMR via formation of iminosulfuranes

    Franz, J.A.; Linehan, J.C.; Lamb, C.N.

    1992-08-01

    We have synthesized new iminosulfuranes from a variety of diaryl-and dialkyl sulfides and dibenzothiophene. The pattern of [sup 15]N chemical shifts indicates that functional groups attached to sulfur are not simply resolved into aryl and alkyl groups. Thus, resolution of sulfur functional groups using [sup 15]N NMR via iminosulfurane does not appear practicable. However, iminosulfurane formation, together with the N-haloamide reaction and the Pummerer rearrangement, provides pathways for chemical discrimination of different sulfur substituents using unique [sup 15]N- or, [sup 19]F-labelled fragments for different categories of sulfur functional groups. In efforts currently underway, we are applying these reactions to methylated extracts and conversion products of the high-organic-sulfur containing Yugoslavian Rasa and Spanish Mequinenza lignites. 1 tab, 14 refs.

  15. Detection of organic sulfur by {sup 15}N and {sup 19}F NMR via formation of iminosulfuranes

    Franz, J.A.; Linehan, J.C.; Lamb, C.N.

    1992-08-01

    We have synthesized new iminosulfuranes from a variety of diaryl-and dialkyl sulfides and dibenzothiophene. The pattern of {sup 15}N chemical shifts indicates that functional groups attached to sulfur are not simply resolved into aryl and alkyl groups. Thus, resolution of sulfur functional groups using {sup 15}N NMR via iminosulfurane does not appear practicable. However, iminosulfurane formation, together with the N-haloamide reaction and the Pummerer rearrangement, provides pathways for chemical discrimination of different sulfur substituents using unique {sup 15}N- or, {sup 19}F-labelled fragments for different categories of sulfur functional groups. In efforts currently underway, we are applying these reactions to methylated extracts and conversion products of the high-organic-sulfur containing Yugoslavian Rasa and Spanish Mequinenza lignites. 1 tab, 14 refs.

  16. Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

    Methods are described to correlate aromatic 1Hδ2/13Cδ2 or 1Hε1/15Nε1 with aliphatic 13Cβ chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with β-carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine δ-CH and tryptophan ε-NH groups

  17. Nitrogen input 15N-signatures are reflected in plant 15N natural abundances of N-rich tropical forest in China

    Abdisa Gurmesa, Geshere; Lu, Xiankai; Gundersen, Per; Yunting, Fang; Mo, Jiangming

    2016-04-01

    In this study, we tested the measurement of natural abundance of 15N15N) for its ability to assess changes in N cycling due to increased N deposition in two forest types; namely, an old-growth broadleaved forest and a pine forest, in southern China. We measured δ15N values of inorganic N in input and output fluxes under ambient N deposition, and N concentration and δ15N of major ecosystem compartments under ambient and increased N deposition. Our results showed that N deposition to the forests was 15N-depleted, and was dominated by NH4-N. Plants were 15N-depleted due to imprint from the 15N-depleted atmospheric N deposition. The old-growth forest had larger N concentration and was more 15N-enriched than the pine forest. Nitrogen addition did not significantly affect N concentration, but it significantly increased δ15N values of plants, and slightly more so in the pine forest, toward the 15N signature of the added N in both forests. The result indicates that the pine forest may rely more on the 15N-depleted deposition N. Soil δ15N values were slightly decreased by the N addition. Our result suggests that ecosystem δ15N is more sensitive to the changes in ecosystem N status and N cycling than N concentration in N-saturated sub-tropical forests.

  18. Isotopic enrichment of 15N by ionic exchange cromatography

    The ionic exchange chromatographic method in columns of resin which is employed in the study of isotopic enrichment of 15N is presented. Determinations are made of the isotopic separation constant for the exchange of isotopes 15N and 14N in the equilibrium involving ammonium hidroxide in the solution phase and ions NH4+ adsorbed in cationic resins: Dowex 50W-X8 and X12, 100-200 mesh. Experiments are also conducted for determination of height of theoretical plates for situations of equilibrium of the NH4+ band in two systems of resin's columns aimed at estimating the experimental conditions used. The isotopic analyses of nitrogen are carried out by mass spectrometry

  19. Marking Drosophila suzukii (Diptera: Drosophilidae) With Rubidium or 15N.

    Klick, J; Yang, W Q; Bruck, D J

    2015-06-01

    Drosophila suzukii Matsumura (Diptera: Drosophilidae) has caused significant economic damage to berry and stone fruit production regions. Markers that are systemic in plants and easily transferred to target organisms are needed to track D. suzukii exploitation of host resources and trophic interactions. High and low concentrations of the trace element, rubidium (Rb), and the stable isotope, 15N, were tested to mark D. suzukii larvae feeding on fruits of enriched strawberry plants grown in containers under greenhouse conditions. Fly marker content and proportion of flies marked 1, 7, and 14 d after emergence from enriched fruits and fly dry mass were analyzed. Nearly 100% of the flies analyzed 14 d after emerging from 15N-enriched plants were marked, whereas only 30-75% and 0-3% were marked 14 d after emerging from high and low Rb concentration plants, respectively. Rapid Rb decay, strong 15N persistence, and the economics of using these markers in the field to elucidate D. suzukii pest ecology are discussed. PMID:26470275

  20. Geomorphic control on the δ15N of mountain forests

    R. G. Hilton

    2013-03-01

    Full Text Available Mountain forests are subject to high rates of physical erosion which can export particulate nitrogen from ecosystems. However, the impact of geomorphic processes on nitrogen budgets remains poorly constrained. We have used the elemental and isotopic composition of soil and plant organic matter to investigate nitrogen cycling in the mountain forest of Taiwan, from 24 sites with distinct geomorphic (topographic slope and climatic (precipitation, temperature characteristics. The organic carbon to nitrogen ratio of soil organic matter decreased with soil 14C age, providing constraint on average rates of nitrogen loss using a mass balance model. Model predictions suggest that present day estimates of nitrogen deposition exceed contemporary and historic nitrogen losses. We found ∼6‰ variability in the stable isotopic composition (δ15N of soil and plants which was not related to soil 14C age or climatic conditions. Instead, δ15N was significantly, negatively correlated with topographic slope. Using the mass balance model, we demonstrate that the correlation can be explained by an increase in nitrogen loss by non-fractioning pathways on steeper slopes, where physical erosion most effectively removes particulate nitrogen. Published data from forests on steep slopes are consistent with the correlation. Based on our dataset and these observations, we hypothesise that variable physical erosion rates can significantly influence soil δ15N, and suggest particulate nitrogen export is a major, yet underappreciated, loss term in the nitrogen budget of mountain forests.

  1. Absorption of ammonium sulphate 15N by coffee plants

    The objective of this study was to quantify the absorption of ammonium sulphate 15N by coffee plants. Treatments consisted of five sub-plots of 9 plants, of which the three central ones received 280 kg ha-1 of 15N, applied at four times: 1/4 on 01 Set 03; 1/4 on 03 Nov 03; 1/4 on 15 Dec 03 and 1/4 on 30 Jan 04. The isotopic enrichment was 2,072 ± 0,001 atom % 15N. The dry matter of the shoot was evaluated every 60 days, using one plant per replicate, collected outside the sub-plot. They were as similar as possible to the labeled plants, which were used only for isotopic and Total N analysis, after being dried at 65 deg C until constant weight. At harvest, plants had absorbed 42,88% of the fertilizer N. Leaves accumulated the largest amount of fertilizer N, and were also the compartments that received most N from other parts of the plant. The following partition of the fertilizer N was found at harvest: 23.01% in young leaves, 6.23% in old leaves, 4,46% in stem, 3.46% in fruits, 3.10% in young branches and 2.63% in old branches. (author)

  2. Variation of natural sup 15 N abundance of crops and soils in Japan with special reference to the effect of soil conditions and fertilizer application

    Yoneyama, Tadakatsu (National Agriculture Research Center, Tsukuba, Ibaraki (Japan). Dept. of Soils and Fertilizers); Kouno, Kazumi; Yazaki, Jinya

    1990-12-01

    The natural {sup 15}N abundance ({delta}{sup 15}N) of the crops subjected to long-term fertilizer treatments under paddy and upland conditions in the different experimental stations throughout Japan were analyzed. The {delta}{sup 15}N values of the grains of paddy rice which were +6.3 per mille on the average in the fields without application of chemical fertilizers decreased by the treatment with chemical fertilizers. The average {delta}{sup 15}N values of the upland crops were lower than those of paddy rice without application of N fertilizers. The {delta}{sup 15}N values of upland crops decreased with the dose of chemical fertilizer N, but increased with the application of composts containing animal feces. The pot experiments using three soils showed that the {delta}{sup 15}N values of paddy rice were higher than those of upland rice and sorghum and that these values were comparable to the {delta}{sup 15}N values of ammonium and nitrate produced in the incubated soils, respectively. The {delta}{sup 15}N values of fertilizer N absorbed by paddy rice were higher than those of fertilizer N, whereas the {delta}{sup 15}N values of the fertilizer N in upland rice and sorghum were increased in the alluvial soils but decreased in Andosols as compared to those of fertilizer N applied. The {delta}{sup 15}N values of the Andosols in Japan showed small variations, with an average value of +6.5 per mille, whereas those of alluvial soils in Japan showed large variations with an average value lower than that of Andosols. (author).

  3. Variation of natural 15N abundance of crops and soils in Japan with special reference to the effect of soil conditions and fertilizer application

    The natural 15N abundance (δ15N) of the crops subjected to long-term fertilizer treatments under paddy and upland conditions in the different experimental stations throughout Japan were analyzed. The δ15N values of the grains of paddy rice which were +6.3 per mille on the average in the fields without application of chemical fertilizers decreased by the treatment with chemical fertilizers. The average δ15N values of the upland crops were lower than those of paddy rice without application of N fertilizers. The δ15N values of upland crops decreased with the dose of chemical fertilizer N, but increased with the application of composts containing animal feces. The pot experiments using three soils showed that the δ15N values of paddy rice were higher than those of upland rice and sorghum and that these values were comparable to the δ15N values of ammonium and nitrate produced in the incubated soils, respectively. The δ15N values of fertilizer N absorbed by paddy rice were higher than those of fertilizer N, whereas the δ15N values of the fertilizer N in upland rice and sorghum were increased in the alluvial soils but decreased in Andosols as compared to those of fertilizer N applied. The δ15N values of the Andosols in Japan showed small variations, with an average value of +6.5 per mille, whereas those of alluvial soils in Japan showed large variations with an average value lower than that of Andosols. (author)

  4. COVALENT BINDING OF REDUCED METABOLITES OF [15N3] TNT TO SOIL ORGANIC MATTER DURING A BIOREMEDIATION PROCESS ANALYZED BY 15N NMR SPECTROSCOPY. (R826646)

    Evidence is presented for the covalent binding ofbiologically reduced metabolites of 2,4,6-15N3-trinitrotoluene(TNT) to different soil fractions (humic acids, fulvicacids, and humin) using liquid 15N NMR spectroscopy. Asilylation p...

  5. A novel method for trapping and analyzing 15N in NO for tracing NO sources

    Kang, Ronghua; Mulder, Jan; Dörsch, Peter

    2016-04-01

    15N isotope tracing is an effective and direct approach to investigate the biological and chemical sources of nitric oxide (NO) in soil. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone. Various chemical conversions of NO to the more stable solutes nitrite (NO2-) and nitrate (NO3-) have been proposed, which allow analysing the 15N abundance without major fractionation. However, NO emissions from soils are usually small, posing major challenges to conversion efficiency and background contamination. Here we present a novel method in which NO is oxidized to NO2- by chromium trioxide (CrO3) prior to conversion to NO2- and NO3- in an alkaline hydrogen peroxide (H2O2) solution. Immediately following trapping, manganese dioxide (MnO2) and 5M HCl are added to remove excess H2O2, and to adjust the pH to around 6.0-7.0, respectively. The resulting solution can be stored until analysis and is none-toxic, allowing to use a modified denitrifier method (Zhu et al., submitted), where NO2- and NO3- are reduced quantitatively to nitrous oxide (N2O). Optimum NO conversion rates of > 90% even at extremely low initial NO concentration were obtained with 4% H2O2, 0.5 M NaOH, and 0.5 L min-1 gas flow rate. In a laboratory test, using NO gas with different 15N signals produced from unlabelled and labelled NO2-, we found an overall precision of 0.4‰ for unlabelled and 49.7‰ for NO enriched with 1.0 atom% 15N, respectively. This indicates that this method can be used for both natural abundance studies of NO, as well as in labelling studies tracing NO sources. Zhu J, Yu L, Bakken LR, Mørkved PT, Mulder J, Dörsch P. Controlled induction of denitrification in Pseudomonas aureofaciens: a modified denitrifier method for 15N and 18O analysis in NO3- from natural water samples by IRMS. Submitted.

  6. Dynamic of N fertilizers: urea (15 N) and aqua ammonia (15 N) incorporated to the sugar cane soil. Final report

    The dynamic of N fertilizers, urea and aqua ammonia, in the soil of sugar cane crops are studied with an emphasis on the horizontal and vertical moving. The nitrogen routing from urea and aqua ammonia sources, by isotopic technique with 15 N in relation to the leaching, volatilization and extraction by the cultivation and residue of N immobilized manure in the soil with sugar cane plantation is also analysed. (C.G.C.)

  7. Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

    Masse, J.E.; Bortmann, P; Dieckmann, T.; Feigon, J

    1998-01-01

    The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,...

  8. /sup 15/N/sub 2/ incorporation and acetylene reduction by azospirillum isolated from rice roots and soils

    Nayak, D.N.; Charyulu, P.B.B.; Rajaramamohan Rao, V. (Central Rice Research Inst., Cuttack (India). Dept. of Soil Microbiology)

    1981-01-01

    Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by /sup 15/N/sub 2/ incorporation and C/sub 2/H/sub 2/ reduction. C/sub 2/H/sub 2/ reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in /sup 15/N/sub 2/ incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N/sub 2/-fixing ability and that plant genotype is of importance for optimal associations.

  9. Recent advances in the application of 13C and 15N NMR spectroscopy to soil organic matter studies

    Nuclear magnetic resonance (NMR) spectroscopy has been applied to many studies in soil science, geochemistry, and environmental science. In recent years, the study of soil organic matter (SOM) using NMR techniques has progressed rapidly. NMR spectroscopy has been used to study chemical changes of SOM during decomposition, and also of soil extract fractions such as humic acid and fulvic acid. NMR spectroscopy of soils has improved rapidly in recent years with the introduction of pre-treatment and particle-size fractionation. In addition to routine liquid- and solid-state 13C NMR applications, 15N NMR spectra of natural abundant samples have been reported, but 15N-enriched material is more convenient to use due to the low natural abundance of 15N. Some newly developed NMR techniques have also been utilised, such as 2-dimensional NMR spectroscopy and improved 1H NMR techniques. These are reviewed and commented on in this paper. Copyright (2000) CSIRO Publishing

  10. Orientation-dependent backbone-only residue pair scoring functions for fixed backbone protein design

    Bordner Andrew J

    2010-04-01

    Full Text Available Abstract Background Empirical scoring functions have proven useful in protein structure modeling. Most such scoring functions depend on protein side chain conformations. However, backbone-only scoring functions do not require computationally intensive structure optimization and so are well suited to protein design, which requires fast score evaluation. Furthermore, scoring functions that account for the distinctive relative position and orientation preferences of residue pairs are expected to be more accurate than those that depend only on the separation distance. Results Residue pair scoring functions for fixed backbone protein design were derived using only backbone geometry. Unlike previous studies that used spherical harmonics to fit 2D angular distributions, Gaussian Mixture Models were used to fit the full 3D (position only and 6D (position and orientation distributions of residue pairs. The performance of the 1D (residue separation only, 3D, and 6D scoring functions were compared by their ability to identify correct threading solutions for a non-redundant benchmark set of protein backbone structures. The threading accuracy was found to steadily increase with increasing dimension, with the 6D scoring function achieving the highest accuracy. Furthermore, the 3D and 6D scoring functions were shown to outperform side chain-dependent empirical potentials from three other studies. Next, two computational methods that take advantage of the speed and pairwise form of these new backbone-only scoring functions were investigated. The first is a procedure that exploits available sequence data by averaging scores over threading solutions for homologs. This was evaluated by applying it to the challenging problem of identifying interacting transmembrane alpha-helices and found to further improve prediction accuracy. The second is a protein design method for determining the optimal sequence for a backbone structure by applying Belief Propagation

  11. Isotope 15N for agronomic research: an overview

    Fertilizer N recovery determined by isotope labelling technique using 15N enriched fertilizer was compared with apparent recovery of N obtained by the difference method and the extent of error associated with it was compared in six vegetable crops. In the difference method, fertilizer N recovery was overestimated and the error ranged from 3 per cent in tomato to 94 per cent in chilli, whereas uptake of soil N by the difference method was underestimated and the error ranged from 2 per cent in tomato to 64 per cent in chilli. One of the main reasons for the error was the degree of response to N due to increase in dry matter yield

  12. Theoretical study of the effective chemical shielding anisotropy (CSA) in peptide backbone, rating the impact of CSAs on the cross-correlated relaxations in L-alanyl-L-alanine

    Benda, Ladislav; Bouř, Petr; Müller, N.; Sychrovský, Vladimír

    2009-01-01

    Roč. 113, č. 15 (2009), s. 5273-5281. ISSN 1520-6106 R&D Projects: GA AV ČR IAA400550701; GA AV ČR IAA400550702; GA MŠk MEB060705 Institutional research plan: CEZ:AV0Z40550506 Keywords : chemical shielding anisotropy * CSA * L-alanyl-L-alanine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.471, year: 2009

  13. An electron-scattering study of 15N

    An electron scattering experiment on 15N was performed in order to test the results of two different shell-model approaches, both performed in a full (0+2)ℎω space, one employing a phenomenologic interaction which is valid throughout the 1p shell, the other an interaction whose parameters were adjusted to fit the excitation energies of a number of states. The experiment was carried out at the high-energy electron-scattering facility of NIKHEF-k. A room temperature gas target was employed. Data were taken at forward angles in the range q=0.35 - 3.17 fm-1. Results are presented for negative-parity states up to an excitation energy of 13 MeV. The differences in groundstate charge density between 15N and the neighbouring nuclei 16O and 14N are compared with results of shell-model calculations. In ch. 5 the transition charge-densities to the excited negative-parity states are presented and compared with shell model calculations. 52 refs.; 18 figs.; 5 tabs

  14. Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

    α-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalytic mechanism, structure-function relationships, and more recently for studies of pro region-assisted protein folding. We have assigned the backbones of the enzyme alone, and of its complex with the tetrahedral transition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, using double- and triple-resonance 3D NMR spectroscopy on uniformly15N- and 13C/15N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibited form of the protein are correlated with distance from the inhibitor, the displacement of backbone nitrogens, and change in hydrogen bond strength upon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzyme with that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), and in a majority of cases the broadening was reversed upon addition of the inhibitor. Implications and possible mechanisms of this line broadening are discussed

  15. Net neutrality at internet backbone provider level

    Baglioni, Laura; Calabrese, Armando; Ghiron, Nathan Levialdi

    2013-01-01

    This paper analysis the Internet interconnection market and combine the main technical (i.e. service quality) and economic aspects (i.e. profits and utility) characterizing relations between market players (end users, EUs; Internet Service Providers, ISPs; Internet Backbone Providers, IBPs) in order to determine possible economic outcomes in the strategic interaction between them. The proposed model enables a comparison to be made between expected values of social welfare (i.e. EU utility and...

  16. Hash: a program to accurately predict protein H{sup {alpha}} shifts from neighboring backbone shifts

    Zeng Jianyang, E-mail: zengjy@gmail.com [Tsinghua University, Institute for Interdisciplinary Information Sciences (China); Zhou Pei [Duke University Medical Center, Department of Biochemistry (United States); Donald, Bruce Randall [Duke University, Department of Computer Science (United States)

    2013-01-15

    Chemical shifts provide not only peak identities for analyzing nuclear magnetic resonance (NMR) data, but also an important source of conformational information for studying protein structures. Current structural studies requiring H{sup {alpha}} chemical shifts suffer from the following limitations. (1) For large proteins, the H{sup {alpha}} chemical shifts can be difficult to assign using conventional NMR triple-resonance experiments, mainly due to the fast transverse relaxation rate of C{sup {alpha}} that restricts the signal sensitivity. (2) Previous chemical shift prediction approaches either require homologous models with high sequence similarity or rely heavily on accurate backbone and side-chain structural coordinates. When neither sequence homologues nor structural coordinates are available, we must resort to other information to predict H{sup {alpha}} chemical shifts. Predicting accurate H{sup {alpha}} chemical shifts using other obtainable information, such as the chemical shifts of nearby backbone atoms (i.e., adjacent atoms in the sequence), can remedy the above dilemmas, and hence advance NMR-based structural studies of proteins. By specifically exploiting the dependencies on chemical shifts of nearby backbone atoms, we propose a novel machine learning algorithm, called Hash, to predict H{sup {alpha}} chemical shifts. Hash combines a new fragment-based chemical shift search approach with a non-parametric regression model, called the generalized additive model, to effectively solve the prediction problem. We demonstrate that the chemical shifts of nearby backbone atoms provide a reliable source of information for predicting accurate H{sup {alpha}} chemical shifts. Our testing results on different possible combinations of input data indicate that Hash has a wide rage of potential NMR applications in structural and biological studies of proteins.

  17. Backbone Dynamics of Triple-helical Collagen-like Structure

    Lazarev, Yu.A.; Lazareva, A.V.; Komarov, V.M.

    1999-01-01

    Some details of the backbone dynamics in the collagen-like triple helix is discussed and the role of backbone dynamics in functioning collagen proteins is illustrated. On a series of oligotripeptides synthetic analogs of collagen formation of high-frequency vibrational backbone dynamics and low-frequency nonlinear backbone dynamics upon stepwise elongation of peptide chain have been described using infrared spectroscopy and hydrogen-exchange method. In the fully completed triple helix the lev...

  18. Natural abundance of /sup 15/N in soil organic matter with special reference to paddy soils in Japan. Biogeochemical implications on the nitrogen cycle

    Wada, Eitaro; Imaizumi, Reiko (Mitsubishi Chemical Industries Ltd., Tokyo (Japan)); Takai, Yasuo

    1984-01-01

    In order to explain the general principle which controls the /sup 15/N content of soil organic nitrogen, experimental rice fields which were fertilized for long term were investigated. The /sup 15/N abundance values of rice plants vary according to the kinds of fertilizer, e.g., chemical fertilizer and green manure. The significant difference of /sup 15/N abundance was recognized between rice plants and fertilizers. Nitrification and denitrification seemed to be responsible for the difference. But these have minor effect on the variation of /sup 15/N abundance of soil because rice plants assimilate all available nitrogen and are removed from paddy fields by harvest. Consequently, the effects of nitrification-denitrification and ammonia volatilization are observed only in biological process such as the growth of rice plants and hydrophytes. A long term addition of fertilizers clearly increased the amount of soil organic nitrogen in paddy fields. Based on isotope mass balance, nitrogen isotope fractionation factor of 0.9942 was estimated in the process of epidiagenesis which indicates the selective decomposition of heavy isotopic species. An addition of ammonium sulfate with low /sup 15/N abundance decreased the /sup 15/N content of soil nitrogen in the paddy fields with the depletion of soil organic nitrogen. OrgC/clay ratio was demonstrated as an important factor which controls the /sup 15/N abundance value in paddy soil. The relation between the /sup 15/N abundance value in soil organic nitrogen and OrgC/clay ratio was able to be presented by hyperbola for the paddy soild so far examined. The /sup 15/N abundance of source and plant nitrogen two isotopic fractionation associated with the epidiagenesis of soil organic matter and the adsorption of ammonia by clay minerals are the three major factors determining the /sup 15/N abundance of soil organic nitrogen.

  19. Application of δ13C and δ15N isotopic signatures of organic matter fractions sequentially separated from adjacent arable and forest soils to identify carbon stabilization mechanisms

    Kayler, Z.E.; Kaiser, M; Gessler, A.; Ellerbrock, R. H.; M. Sommer

    2011-01-01

    Identifying the chemical mechanisms behind soil carbon bound in organo-mineral complexes is necessary to determine the degree to which soil organic carbon is stabilized belowground. Analysis of δ13C and δ15N isotopic signatures of stabilized OM fractions along with soil mineral characteristics may yield important information about OM-mineral associations and their processing history. We anlayzed the δ13C and δ15N isotopic signatures from two organic matter (OM) fractio...

  20. Effect of fed-batch on synthesis of 15N-L-tryptophan from precursor fermentation

    Using Candida utilis AS60 as 15N-L-tryptophan producing strain, the influence by different feeding modes of glucose, 15N-(NH4)2SO4 and 15N-anthranilic acid was studied. The results of these experiments show that the yield of 15N-L-tryptophan was 3.073 g/L by addition of 50 g/L of glucose, 2.1 g/L of 15N-(NH4)2SO4 and 1.5 g/L of 15N-anthranilic acid after 36 h of fermentation. (authors)

  1. A synthetic HIV-1 subtype C backbone generates comparable PR and RT resistance profiles to a subtype B backbone in a recombinant virus assay.

    David Nauwelaers

    Full Text Available In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE, p24 and Viral Load (VL were monitored. The resulting HIV-1-C recombinant virus stocks (RVS were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii their rate of infection was slower than viruses generated in the subtype B backbone; iii they did not produce clear CPE in MT4 cells; and iv drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.

  2. Toward Improved Description of DNA Backbone: Revisiting Epsilon and Zeta Torsion Force Field Parameters

    Zgarbová, M.; Luque, F. J.; Šponer, Jiří; Cheatham III, Thomas E.; Otyepka, M.; Jurečka, P.

    2013-01-01

    Roč. 9, č. 5 (2013), s. 2339-2354. ISSN 1549-9618 Grant ostatní: GA ČR(CZ) GAP208/10/1742 Institutional support: RVO:68081707 Keywords : MOLECULAR- DYNAMICS SIMULATIONS * PHOSPHORUS CHEMICAL-SHIFTS * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 5.310, year: 2013

  3. Investigation of the metabolism of colostomized laying hens with 15N-labelled wheat. 6

    Three colostomized laving hens received 40 g 15N-labelled wheat with 20.13 atom-% 15N excess (15N'), 19.18 atom-% 15N'-lysine, 18.17 atom-% 15N'-histidine and 20.43 atom-% 15N'-arginine per day over a period of four days. After having received the same non-labelled feed ration on the following four days, the hens were slaughtered. The incorporation and distribution of 15N' in the total nitrogen and the nitrogen of the basic amino acids was determined in liver, kidneys, muscles, bones and the remaining carcass (excluding blood, digestive tract and genital organs). The quota of nitrogen of natural isotope frequency (14N) of the total 14N of the hens' carcasses was 47% in the muscles, 14% in the bones and 20% in the feathers; the relative 15N' values were 37%, 8% and 1%, resp. The atom-% 15N' in the kidneys was twice as much as in the liver four days after the last 15N' application. The average percentage of the nitrogen in the three basic amino acids of the total nitrogen in the tissues and organs (excluding feathers) is 25% concerning both 14N and 15N'. The 15N' balance revealed that in hen 1 100%, in hen 2 102% and in hen 3 101% of the consumed wheat 15N' were found. (author)

  4. SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network

    Shen Yang; Bax, Ad, E-mail: bax@nih.go [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2010-09-15

    NMR chemical shifts provide important local structural information for proteins and are key in recently described protein structure generation protocols. We describe a new chemical shift prediction program, SPARTA+, which is based on artificial neural networking. The neural network is trained on a large carefully pruned database, containing 580 proteins for which high-resolution X-ray structures and nearly complete backbone and {sup 13}C{sup {beta}} chemical shifts are available. The neural network is trained to establish quantitative relations between chemical shifts and protein structures, including backbone and side-chain conformation, H-bonding, electric fields and ring-current effects. The trained neural network yields rapid chemical shift prediction for backbone and {sup 13}C{sup {beta}} atoms, with standard deviations of 2.45, 1.09, 0.94, 1.14, 0.25 and 0.49 ppm for {delta}{sup 15}N, {delta}{sup 13}C', {delta}{sup 13}C{sup {alpha}}, {delta}{sup 13}C{sup {beta}}, {delta}{sup 1}H{sup {alpha}} and {delta}{sup 1}H{sup N}, respectively, between the SPARTA+ predicted and experimental shifts for a set of eleven validation proteins. These results represent a modest but consistent improvement (2-10%) over the best programs available to date, and appear to be approaching the limit at which empirical approaches can predict chemical shifts.

  5. SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network

    NMR chemical shifts provide important local structural information for proteins and are key in recently described protein structure generation protocols. We describe a new chemical shift prediction program, SPARTA+, which is based on artificial neural networking. The neural network is trained on a large carefully pruned database, containing 580 proteins for which high-resolution X-ray structures and nearly complete backbone and 13Cβ chemical shifts are available. The neural network is trained to establish quantitative relations between chemical shifts and protein structures, including backbone and side-chain conformation, H-bonding, electric fields and ring-current effects. The trained neural network yields rapid chemical shift prediction for backbone and 13Cβ atoms, with standard deviations of 2.45, 1.09, 0.94, 1.14, 0.25 and 0.49 ppm for δ15N, δ13C', δ13Cα, δ13Cβ, δ1Hα and δ1HN, respectively, between the SPARTA+ predicted and experimental shifts for a set of eleven validation proteins. These results represent a modest but consistent improvement (2-10%) over the best programs available to date, and appear to be approaching the limit at which empirical approaches can predict chemical shifts.

  6. Backbone Guided Local Search for the Weighted Maximum Satisfiability Problem

    Jiang, He; Xuan, Jifeng

    2009-01-01

    In this chapter, analytical results on the backbone in weighted MAX-SAT were presented in this paper. We showed that it is intractable to retrieve the backbone in weighted MAX-SAT with any performance guarantee under the assumption that P NP . And a backbone guided local search algorithm was proposed for weighted MAX-SAT. Results of this paper imply a new way to incorporate the backbone in heuristics. The approximate backbone used to guide the flipping of literals in those local search based...

  7. Porous solid backbone impregnation for electrochemical energy conversion systems

    Boulfrad, Samir

    2013-09-19

    An apparatus and method for impregnating a porous solid backbone. The apparatus may include a platform for holding a porous solid backbone, an ink jet nozzle configured to dispense a liquid solution onto the porous solid backbone, a positioning mechanism configured to position the ink jet nozzle proximate to a plurality of locations of the porous solid backbone, and a control unit configured to control the positioning mechanism to position the ink jet nozzle proximate to the plurality of locations and cause the ink jet nozzle to dispense the liquid solution onto the porous solid backbone.

  8. Convenient and scalable synthesis of fmoc-protected Peptide nucleic Acid backbone.

    Feagin, Trevor A; Shah, Nirmal I; Heemstra, Jennifer M

    2012-01-01

    The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA. PMID:22848796

  9. Peptoid-Peptide hybrid backbone architectures

    Olsen, Christian Adam

    2010-01-01

    Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with beta-peptides and the so-called peptoids (N-alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both alpha- and beta......-amino acids (alpha/beta-peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of alpha-amino acids and peptoids, including beta-peptoids (N-alkyl-beta-alanine oligomers), and is intended to give an overview of this area...

  10. Understanding VoIP from Backbone Measurements

    Birke, Robert Rene' Maria; Petracca, Michele; Mellia, Marco

    2007-01-01

    VoIP has widely been addressed as the technology that will change the Telecommunication model opening the path for convergence. Still today this revolution is far from being complete, since the majority of telephone calls are originated by circuit-oriented networks. In this paper for the first time to the best of our knowledge, we present a large dataset of measurements collected from the FastWeb backbone, which is one of the first worldwide Telecom operator to offer VoIP and high-speed data ...

  11. Determination of 15N nitrates in water samples using mass spectrometry

    The nitrogen element (Z = 7) has two stable isotopes, whose relative quantities are 99.64% for 14N and 0.36% for 15N. Nitrogen is part of many processes and reactions that are important to life and that affect the quality of the water. Within the nitrogen cycle there are kinetic and thermodynamic fractionation processes, which are potentially important for tracing its sources and demands. Water contamination due to nitrates is a serious problem that is affecting large parts of the biosphere. Surface water contamination can be remedied by prevention and control measures, but the problem becomes acute when the contamination penetrates to groundwater water. Contaminated groundwater can remain in the aquifers for centuries, even milleniums, and decontamination is very difficult, if not impossible. Isotopic techniques can help to evaluate how vulnerable the groundwater is to contamination from the surface when its displacement speed and extra load area are determined. Then the sources of surface contamination (natural, industrial, agricultural, domestic) can be identified. Isotopic techniques can also describe an incipient contamination, and they can provide an early alert when chemical or biological indicators do not reveal any signs for concern. The isotopic fractionation of several nitrogen compounds provide the basis for using 15N as a hydrological isotope tool. There are three main sources of nitrogen contamination in water, these are: organic nitrogen in the soil, nitrogenized fertilizers, domestic, industrial and animal wastes. The following technical procedure describes the method for determining the isotopic ration 15N/14N in nitrates in water. The nitrate is separated from the water using ion exchange columns through a resin, which is eluded with HCI and with the addition of silver oxide becomes silver nitrate. This solution is freeze-dried and submitted to combustion at 850 in a sealed quartz tube, using copper/copper oxide for the nitrogen reduction and

  12. Use of Bio-Organic Fertilizers to Develop N Uptake Using 15N Technique

    Experimental work either in field scale or in green house conditions were conducted using 15N technique to evaluate the role of different bio fertilizers and different plant residues as organic amendments on enhancement of plant N nutrition. Nitrogen fixation by a symbiotic bacteria has been observed in greenhouse and field experiments under dry land cropping systems. Biological N2 fixation associated with crop residues (legumes or cereals) was investigated in pot experiments with wheat and chickpea cultivars. In these experiments, labelled wheat and rice straw were used as organic N sources in comparison with either 15N-labelled ammonium sulfate or ammonium nitrate as chemical nitrogen fertilizers. Rhizobium inoculation extended to be used with wheat gave the best results of N uptake and N2 fixation when combined with Azospirillum brasilense as heterotrophic diazotrophs. The nitrogen uptake by wheat plants was significantly increased by application of soybean residues and inoculation with Azospirillum brasilense. From the field trial we can conclude that soybean residue as enriched N material, and Azospirillum brasilense inoculation enhanced N yields of wheat cultivars grown in poor fertile sandy soil

  13. Impact of Backbone Fluorination on π-Conjugated Polymers in Organic Photovoltaic Devices: A Review

    Nicolas Leclerc

    2016-01-01

    Full Text Available Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone.

  14. Metabolic studies in colostomized laying hens using 15N-labelled wheat. 4

    3 colostomized laying hybrids received over 4 days a dosage of 672 mg 15N excess (15N'), 20.3 mg lysine 15N', 23.0 mg histidine 15N' and 66.7 mg arginine 15N' with a ration customary in production. After feeding the same unlabelled ration for another 4 days the hens were killed and the N content of the blood as well as of its fractions (cells, plasma, free amino acids of the plasma) was determined. The 15N' was determined in the total blood, the corpuscles, the plasma, the nonprotein-N (NPN) fraction as well as in the amino acids lysine, histidine and arginine. The average amount of the blood cell N in the total blood N was 58.5% and that of the plasma 40.3%; the corresponding 15N' values amounted to 66.1% and 33.9%, respectively. The sum of the 15N' of the basic amino acids of the blood cells, on an average, amounted to 39.7% of the total cell 15N'; the corresponding average value for the total 15N' in lysine, histidine and arginine of the blood plasma 15N' was 23.6.% and the quota of the three free amino acids of the total NP15N' of the plasma was 6.2%. (author)

  15. Utilization of natural variations in the isotopic abundance of 15N to trace the source of aquifer pollution by nitrates

    The validity of using the natural isotope nitrogen-15 to trace the source of nitrates contained in aquifers is discussed with reference to experimental devices (lysimeters and experimental plots) and for examples chosen from the Paris area. There are a number of sources of nitrates: (1) industrially synthesized nitrates (fertilizers); (2) nitrates produced by oxidation of organic matter associated with human, agricultural or urban activities; (3) nitrates synthesized in the soil by the decay of organic matter. In the examples studied these sources differ in their 15N content: (1) fertilizers have a delta15N close to zero (atmospheric nitrogen); (2) the nitrates originating from organic pollution have high delta15N (above 10-12 per mille) and this 15N enrichment is associated with the volatilization of ammonia during the ammonia stage of mineralization; (3) the isotopic characterization of the nitrates produced by organic matter in the soil is less evident. Citing several examples, the author demonstrates that these three sources are diffentiated isotopically. Consideration of the parameters nitrate concentration/isotopic composition reveals simple mixture curves. In the most complicated cases - where there is association with other isotopic (3H) or chemical parameters - it is possible qualitatively to trace the sources of nitrogen pollution

  16. FAO/IAEA - interregional training course on the use of 15N in soil science and plant nutrition

    This training manual provides an introduction for the basic methodology and principles of application of the stable isotope 15N. After preliminary remarks on stable isotope terminology fundamentals, experimental problems and methods of quantitative nitrogen determination in soil and plant studies are reported in the main part of the manual. An appendix with a compilation of different parameters such as natural abundance of stable isotopes, selected atomic weights and multiples of them conversion factors of chemical compounds, and much more concludes the manual

  17. Mechanism of Solid-State Thermolysis of Ammonia Boraine: 15N NMR Study Using Fast Magic-Angle Spinning and Dynamic Nuclear Polarization

    Kobayashi, Takeshi [Ames Laboratory; Gupta, Shalabh [Ames Laboratory; Caporini, Marc A [Bruker BioSpin Corporation; Pecharsky, Vitalij K [Ames Laboratory; Pruski, Marek [Ames Laboratory

    2014-08-28

    The solid-state thermolysis of ammonia borane (NH3BH3, AB) was explored using state-of-the-art 15N solid-state NMR spectroscopy, including 2D indirectly detected 1H{15N} heteronuclear correlation and dynamic nuclear polarization (DNP)-enhanced 15N{1H} cross-polarization experiments as well as 11B NMR. The complementary use of 15N and 11B NMR experiments, supported by density functional theory calculations of the chemical shift tensors, provided insights into the dehydrogenation mechanism of AB—insights that have not been available by 11B NMR alone. Specifically, highly branched polyaminoborane derivatives were shown to form from AB via oligomerization in the “head-to-tail” manner, which then transform directly into hexagonal boron nitride analog through the dehydrocyclization reaction, bypassing the formation of polyiminoborane.

  18. Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

    Tunlid, A.; Odham, G.; Findlay, R. H.; White, D. C.

    1985-01-01

    Sensitive detection of cellular components from specific groups of microbes can be utilized as 'signatures' in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacterial) cell wall, can be detected reproducibly. Enrichments of D-[15N]alanine determined in E. coli grown with [15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M - HF)- and (M - F or M + H - HF)- formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10(3)-10(4) cells, as a function of the 15N-14N ratio.

  19. Fate of 15N-urea and 15N-ammonium sulphate applied in different periods to cica-8 rice culture in greenhouse conditions

    The fate of nitrogen fertilizers in rice cultivars (Cica-8) is studied. Urea (1.973% at of 15N) and ammonium sulfate (1.826% at of 15N) are used. The fertilizers are applied in four levels (0,100,200 and 300 Kg N/ha) in shadow coditions and after 30 days of germination. (M.A.C.)

  20. Utilization of 15N-labelled urea in laying hens. 4

    In order to study the utilization of urea in poultry, 3 colostomized laying hybrids were orally supplied with a traditional ration supplemented with 1% 15N'-labelled urea with a 15N excess (15N') of 96.06 atom-% over a period of 6 days. After another 2 days on which the hens received the same ration with unlabelled urea, they were killed. The atom-% 15N' of the blood on an average of the 3 hens was 0.64, of the plasma 1.40 and of the corpuscles 0.47. The TCA-soluble fraction of the blood had an average 15N' of 1.14 atom-%; the 15N amount was 9.7% of the total amount of 15N in the blood. The amount of 15N' in the urea in the blood was 6.8 atom-%. This shows that the absorbed urea is decomposed very slowly. The quota of 15N' in the basic amino acids from the total 15N' of the blood plasma was only 0.3% and that of the corpuscles 2.2%. The average 15N' of the mature follicles was 2.39 atom-% whereas the smallest and the remaining ovary contain 1.12 atom-%. The labelling level of lysine in mature egg cells was, in contrast to this, only 0.08 atom-% 15N' and in infantile follicles 0.04 atom-% 15N'. 1% of the 15N' quota was in the follicles and the remaining ovary. Of the basic amino acids, histidine is most strongly labelled. The lower incorporation of the 15N' from urea into the basic amino acids shows that the nitrogen of this compound can be used for the synthesis of the essential amino acids to a low degree only. (author)

  1. The influence of DNA binding on the backbone dynamics of the yeast cell-cycle protein Mbp1

    Mbp1 is a transcription factor involved in the regulation of the cell cycle in yeast. The N-terminus of this protein contains a DNA binding domain that includes a winged helix-turn-helix motif. The C-terminal 24 residues of this domain (the 'tail') are disordered in the crystal state, but are important for DNA binding. We have measured 15N NMR relaxation rates at 11.75 and 14.1 T to determine the dynamics of the free protein and in its complex with a specific DNA duplex. The dynamics data were quantitatively analysed using both spectral density mapping and the Lipari-Szabo formalism including the effects of chemical exchange and rotational anisotropy. A detailed analysis has been made of the effect of anisotropy, exchange and experimental precision on the recovered motional parameters. The backbone NH relaxation is affected by motions on a variety of time scales from millisecond to tens of picoseconds. The relaxation data show a structured core of 100 residues corresponding to that observed in the crystal state. Within the core of the protein, two regions on either side of the putative recognition helix (helix B) show slow (ca. 0.2 ms) conformational exchange dynamics that are quenched upon DNA binding. The C-terminal 24 residues are generally more dynamic than in the core. However, in the free protein, a stretch of ∼8 residues in the middle of the tail show relaxation behaviour similar to that in the core, indicating a structured region. NOEs between Ala 114 in this structured part of the tail and residues in the N-terminal beta strand of the core of the protein demonstrate that the tail folds back onto the core of the protein. In the complex with DNA, the structured part of the tail extends by ca. 3 residues. These data provide a framework for understanding the biochemical data on the mechanism and specificity of DNA binding

  2. Backbone and side-chain 1H, 13C and 15N assignments of the ubiquitin-associated domain of human X-linked inhibitor of apoptosis protein

    Hui, Sin-Kam; Tse, Man-Kit; Yang, Yinhua; Wong, Benjamin Chun-Yu; Sze, Kong-Hung

    2010-01-01

    X-linked inhibitor of apoptosis protein (XIAP), a leading member of the family of inhibitor of apoptosis (IAP) proteins, is considered as the most potent and versatile inhibitor of caspases and apoptosis. It has been reported that XIAP is frequently overexpressed in cancer and its expression level is implicated in contributing to tumorigenesis, disease progression, chemoresistance and poor patient-survival. Therefore, XIAP is one of the leading targets in drug development for cancer therapy. ...

  3. The synthesis of barbituric acid and some of its derivatives isotopically labelled with 15N

    Full text: Barbituric acid is the parent compound of a large class of barbiturates that have central nervous system depressant properties, although barbituric acid itself is not pharmacologically active. In recent years, barbituric acid derivatives have been studied as antitumor, anticancer and anti-osteoporosis agents. The aim of this paper is to present the synthesis of barbituric acid-15N, 5,5-diethylbarbituric acid-15N (Veronal-15N) and 5-ethyl-5-phenylbarbituric acid- 15N (Phenobarbitone-15N) . As isotopically labelled material we used urea-15N2, 99 at.% 15N produced at National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania. All compounds were fully characterized by Mass Spectrometry analyses, by FT-IR Spectroscopy and RX Diffraction, and the isotopic label was determined by MS on the molecular compounds. (author)

  4. Pion elastic and inelastic scattering from 15N

    Data were obtained on the Clinton P. Anderson Los Alamos Meson Physics Facility Energetic Pion Channel and Spectrometer for elastic and inelastic pion scattering from ground state 15N nuclei. States observed here included those of 0.0, 5.27, 6.32, 7.16, 7.30, 7.57, 8.31, 8.57, 9.15, 9.76, 9.9, 10.7, 11.3, 11.9, 12.5, 12.9, 13.1, 14.1, 14.4, 14.6, 15.0, 16.5, 16.9, 17.2, 17.6, 18.3, 18.7, and 18.9 MeV excitation energies. Angular distributions were obtained for scattering at angles from 25 degree to 90 degree in 5 degree increments with an incident pion energy of 164 MeV. Optical model analyses of the elastic (0 MeV) angular distributions with equal point proton and neutron densities in both momentum and coordinate space formulations accurately predict the data, although the two formulations require different energy shifts to do so. This difference is thought to be a result of the more accurate nonlocal representation of the nuclear potential in the momentum space code. Additional spectra were obtained for scattering at constant momentum transfers of .94 and 1.57 fm-1 in order to generate constant momentum transfer excitation functions. Use of these excitation functions, σ(π+)/σ(π-) ratios, and shell model DWIA calculations allowed identification of several excited states having shell-model-like, single particle-hole, pure spin-flip excitations. Shell model and collective model DWIA calculations, as well as the q = .94 and 1.57 fm-1 excitation functions and the σ(π+)/σ(π-) ratios indicate that the other states are generally well represented by a shell model description with collective enhancements

  5. Studies with 15N-labelled lysine in colostomized laying hens. 5

    3 colostomized laying hens received, together with a commercial ration of 120 g, 0.2 % 15N-labelled L-lysine with an atom-% 15N excess (15N') of 48 %; subsequently the same ration was fed over a period od 4 days with 0.2 % unlabelled L-lysine. After the end of the experiment the hens were slaughtered. The atom-% 15N' was determined in total, in the lysine, histidine and arginine N of blood cells, plasma, NPN fraction of the blood, stomach, small intestine, cecum and rectum. 15N' in the blood cells was 0.11 atom-% in the blood plasma 0.17 atom-%, in the NPN fraction of the blood 0.09 atom-%, in the tissues of the gastrointestinal tract 0.11 atom-% and in its contents 0.12 atom-%. On the average the blood contained per hen 77.9 % lysine-15N', 16.4 % arginine-15N' and 5.7 % histidine-15N' of the basic amino acid-15N'. For the gastrointestinal tract 78.7 % lysine-15N', 19.0 % arginine-15N' and 2.3 % histidine-15N' of the 15N' of the basic amino acids were ascertained. In comparison to histidine the α-amino-N of lysine is incorporated to a considerably higher degree into arginine. For lysine and arginine the atom-% 15N' in the contents of the gastrointestinal tract is 4 days after the end of the supplementation of labelled lysine 8 to 10 times higher than in the feces of the last day of the experiment. This indicates a considerable secretion of the 2 amino acids in the gastrointestinal tract and their reabsorption to a large extent. (author)

  6. Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: Application to unfolded proteins

    Panchal, Sanjay C.; Bhavesh, Neel S.; Hosur, Ramakrishna V. [Tata Institute of Fundamental Research, Department of Chemical Sciences (India)

    2001-06-15

    Two triple resonance experiments, HNN and HN(C)N, are presented which correlate H{sup N} and {sup 15}N resonances sequentially along the polypeptide chain of a doubly ({sup 13}C, {sup 15}N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C{sup {alpha}} and C{sup {beta}} chemical shift dispersions.

  7. Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: Application to unfolded proteins

    Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resonances sequentially along the polypeptide chain of a doubly (13C, 15N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, Cα and Cβ chemical shift dispersions

  8. 15N tracer kinetic studies on the validity of various 15N tracer substances for determining whole-body protein parameters in very small preterm infants

    Reliable 15N tracer substances for tracer kinetic determination of whole-body protein parameters in very small preterm infants are still a matter of intensive research, especially after some doubts have been raised about the validity of [15N]glycine, a commonly used 15N tracer. Protein turnover, synthesis, breakdown, and further protein metabolism data were determined by a paired comparison in four preterm infants. Their post-conceptual age was 32.2 +/- 0.8 weeks, and their body weight was 1670 +/- 181 g. Tracer substances applied in this study were a [15N]amino acid mixture (Ia) and [15N]glycine (Ib). In a second group of three infants with a post conceptual age of 15N-labeled 32.0 +/- 1.0 weeks and a body weight of 1,907 +/- 137 g, yeast protein hydrolysate (II) was used as a tracer substance. A three-pool model was employed for the analysis of the data. This model takes into account renal and fecal 15N losses after a single 15N pulse. Protein turnovers were as follows: 11.9 +/- 3.1 g kg-1 d-1 (Ia), 16.2 +/- 2.5 g kg-1 d-1 (Ib), and 10.8 +/- 3.0 g kg-1 d-1 (II). We were able to demonstrate an overestimation of the protein turnover when Ib was used. There was an expected correspondence in the results obtained from Ia and II. The 15N-labeled yeast protein hydrolysate is a relatively cheap tracer that allows reliable determination of whole-body protein parameters in very small preterm infants

  9. Use of 15N Label in Organic Synthesis and Spectroscopy. Part I: Preparation of 15N-Labeled tert-Butylamine

    Talaty, Erach R.; Boese, Christopher A.; Adewale, Sanni M.; Ismail, Mohammed S.; Provenzano, Frank A.; Utz, Melissa J.

    2002-02-01

    The preparation of 15N-labeled tert-butylamine involves the conversion of the correspondingly labeled potassium cyanide into the 15N-labeled tert-butylformamide via the Ritter reaction in 85% yield, followed by hydrolysis with either aqueous sodium hydroxide or hydrochloric acid. The NMR spectra of the compounds provide a valuable opportunity for discussing several important topics in NMR spectroscopy, such as cis-trans isomerism due to restricted rotation and 15N coupling. Comparison of the IR spectra of the labeled and unlabeled compounds permits a forum for discussing the theory of vibrational frequencies.

  10. Studies with 15N-labelled lysine in colostomized hens. 3

    In a metabolism experiment with 15N-labelled lysine 3 colostomized laying hybrids received over 4 days 0.2% L-lysine with 48 at% 15N excess (15N') in addition to a ration conventionally produced and, subsequent to this, unlabelled lysine for four days. At the end of the experiment the hens were killed and the individual organs and tissues were prepared for 15N analysis. The incorporation of the lysine-15N' into the further amino acids of follicles, ovary and oviduct is described. The at% 15N' of the complete range of amino acids was analyzed in the individual follicles. Various levels of heavy nitrogen could be detected in all essential and non-essential amino acids. Of the total amount of 15N' detected in the follicles 64.0%, 65.0% and 61.2%, resp., could be detected in lysine and 25.2%, 25.4% and 28.7%, resp., in the other amino acids (hens 1 to 3). In the ovary on average 61.6% and in the oviduct 54.2% of the respective 15N' amount was detected in lysine. In the ovary 10.9% and in the oviduct 8.4% 15N' of the total 15N' of these samples were incorporated into the arginine molecules. (author)

  11. Alanine flux in obese and healthy humans as evaluated by 15N- and 2H3-labeled alanines

    Estimates of plasma alanine flux as measured in humans using L-[15N]-alanine or L-[3,3,3-2H3]alanine were compared by simultaneous intravenous infusion of both tracers. Plasma isotope enrichments were measured by chemical ionization gas chromatography-mass spectrometry. In 16 obese women before and during a hypocaloric diet and in 4 normal men in the postabsorptive and fed states, the fluxes were highly correlated (r2 = 0.93) although plasma alanine flux with the 2H tracer was two to three times greater than that obtained with [15N]alanine. The fluxes decreased with the hypocaloric diet in obese subjects and increased during the fed state in healthy adults. Thus, although the estimates of alanine flux differed according to the tracer used, both appear to give equivalent information about changes in alanine kinetics induced by the nutritional conditions examined

  12. Utilization of 15N-labelled urea in laying hens. 7

    3 colostomized laying hybrids received 1% 15N-labelled urea with 96.06 atom-% 15N excess (15N') with a commercial ration over a period of 6 days. After the application of the same ration with unlabelled urea on the following 2 days the animals were butchered. In the muscles of breast, legs and heart, the labelling of total nitrogen and the incorporation of urea 15N' into 15 amino acids of the 3 different kinds of muscles were ascertained. On average, significant differences could be ascertained between the atom-% 15N of the muscles was 0.25 and 0.34 atom-%, resp.; that of the cardial proteins 0.71 atom-% 15N'. The incorporation of urea 15N into the basic amino acids is low and varies both between the kinds of muscles and between the amino acids. On average the highest level of labelling was found among the essential amino acids valine, isoleucine and leucine; the average atom-% 15N' for the muscles of the breast is 0.13, of the leg 0.17, and of the heart 0.27; the 15N' quota of branched Chain amino acids in the total 15N' of the respective muscle is accordingly 6.0%, 5.0% and 4.5%. The non-essential amino acids, particularly glutamic acid, are more highly labelled in the muscles than the essential ones. A 15N' for glutamic acid of 0.24 atom-% in the breast muscles, of 0.27 atom-% in those of the legs and of 0.64 atom-% in the heart muscle could be detected. The average quota of the 15N' of these acid amino acids in the 15N' for breast, leg and heart muscles is 7.4, 6.2 and 6.7, resp. The quota of the 15N' in the 6 non-essential amino acids in the total 15N' in all 3 kinds of muscles is approximately two thirds and in the 9 essential ones one third of the total 15N'. Although the results show that there is a certain incorporation of 15N' from urea into the amino acids of the muscle proteins, their contribution to meeting the demands is irrelevant. (author)

  13. The Use Of 15N in the Study of Nitrogen Uptake and Metabolism in Plants

    Some forty years ago Mattson attempted to represent soil solutions as ionic states. Later on, he further developed his theory with the aid of the latest achievements in physical chemistry. In 1955 Schoffield applied chemical thermodynamics to make the interrelations between the solid and liquid phases of the soil even more precise. Nitrogen occupies a special position among the plant nutrients. The greatest success in nitrogen uptake and metabolism studies, however, has been achieved only recently after the development of isotope techniques. The study of nitrogen metabolism using isotope techniques has been carried out for some years at the N. Poushkarov Institute of Soil Science using optical methods of isotope detection. Certain of the results obtained recently point to the great opportunities offered by the use of the optical method. Greenhouse and field experiments were carried out with wheat, oats and lucerne. Ammonium sulphate with 11.50 at.%, 15N,andurea 5.55 at % were used as sources of nitrogen. Depending on the conditions, the nitrogen introduced with fertilizers was utilized by the plants in amounts ranging from 47 to 56% in the greenhouses, and from 38 to 45% m the field. It was established that the soil was the source of nearly half the nitrogen of the plants. Fertilized plants took up more of the soil nitrogen than the unfertilized plants. The nitrogen introduced into the soil was found in all fractions of the plants after 24 h and was in the non-protein organic nitrogen, constitution proteins, chlorophyll and reserve proteins of the plants. The highest amounts of 15N were found in the following free amino acids: arginine, histidine, lysine and the amide aspargine. In the bound amino acids, alanine, threonine, serine and glycine were highest in 15N. Phosphorus application increased the amounts of nitrogen in the amino acids. It was established that nitrogen turnover was greatest in chlorophyll and the constitution proteins. In the study of the quality

  14. Protein backbone motions viewed by intraresidue and sequential HN-Hα residual dipolar couplings

    Triple resonance E.COSY-based techniques were used to measure intra-residue and sequential HN-Hα residual dipolar couplings (RDCs) for the third IgG-binding domain of protein G (GB3), aligned in Pf1 medium. Measurements closely correlate with values predicted on the basis of an NMR structure, previously determined on the basis of a large number of one-bond backbone RDCs measured in five alignment media. However, in particular the sequential HN-Hα RDCs are smaller than predicted for a static structure, suggesting a degree of motion for these internuclear vectors that exceeds that of the backbone amide N-H vectors. Of all experimentally determined GB3 structures available, the best correlation between experimental 1H-1H couplings is observed for a GB3 ensemble, previously derived to generate a realistic picture of the conformational space sampled by GB3 (Clore and Schwieters, J Mol Biol 355:879-886, 2006). However, for both NMR and X-ray-derived structures the 1H-1H couplings are found to be systematically smaller than expected on the basis of alignment tensors derived from 15N-1H amide RDCs, assuming librationally corrected N-H bond lengths of 1.041 A

  15. Acetylene inhibition of N2O reduction in laboratory soil and groundwater denitrification assays: evaluation by 15N tracer and 15N site preference of N2O

    Weymann, Daniel; Well, Reinhard; Lewicka-Szczebak, Dominika; Rohe, Lena

    2013-01-01

    Acetylene inhibition of N2O reduction in laboratory soil and groundwaterdenitrification assays: evaluation by 15N tracer and 15N site preference ofN2ODaniel Weymann (1), Reinhard Well (2), Dominika Lewicka-Szczebak (2,3), and Rohe Lena (2)(1) Forschungszentrum Juelich, Agrosphere Institute (IBG-3), Juelich, Germany (), (2)Thünen-Institute of Climate-Smart Agriculture, Braunschweig, Germany, (3) University of Wroclaw, PolandThe measurement of denitrification in soils and...

  16. Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

    A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids

  17. Investigation into endogenous N metabolism in 15N-labelled pigs. 1

    4 male castrated pigs (55-65 kg) either received a wheat-fish meal diet (1 and 2) or a wheat-horse bean diet (3 and 4) without straw meal supplement (1 and 3) or with a supplement of 20% dry matter (2 and 4). In order to investigate whether a 15N labelling of the pigs is also possible with a protein excess in the ration, the animals received 24.8 g (1 and 2) and 11.6 g crude protein/kg/sup 0.75/ live weight (3 and 4). During a 10-day 15N-labelling 385 mg 15N excess (15N') per kg/sup 0.75/ were applied with 15N labelling the following quotas of the applied 15N amount were incorporated: 1 = 10.2%, 2 = 7.2%, 3 = 18.7%, 4 = 14.4%. 15N excretion in both TCA fractions of feces showed a highly significant positive correlation to the increasing content of crude fibre in the 4 diets. The immediate 15N incorporation into the TCA-precipitable fraction of feces proves that 15N enters the large intestine endogenously and serves bacterial protein synthesis. 3 days after the last 15 application the pigs were killed. The values of atom-% 15N' were determined in the TCA-precipitable blood plasma and in the TCA-precipitable fraction of the liver. The other examined organs and tissues showed smaller differences between the test animals. The results show that the 15N labelling of tissues and organs of pigs is also possible at a high level of protein supply by means of an oral application of [15N] ammonia salts. (author)

  18. High Performance Infiltrated Backbones for Cathode-Supported SOFC's

    Gil, Vanesa; Kammer Hansen, Kent

    2014-01-01

    A four-step infiltration method has been developed to infiltrate La0.75Sr0.25MnO3+δ (LSM25) nanoparticles into porous structures (YSZ or LSM-YSZ backbones). The pore size distribution in the backbones is obtained either by using PMMA and/or graphites as pore formers or by leaching treatment of...

  19. Determination of Protein Backbone Structures from Residual Dipolar Couplings

    Prestegard, J H; Mayer, K. L.; Valafar, H.; Benison, G. C.

    2005-01-01

    There are a number of circumstances where a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative where computational modeling of many sequentially related structures from the backbone of a single family representative is anti...

  20. Direct measurement of the 15N CSA/dipolar relaxation interference from coupled HSQC spectra

    Here we propose a method for the measurement of the 15N CSA/dipolar relaxation interference based on direct comparison of the 15N doublet components observed in a 1H-coupled 1H-15N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the 15N CSA/dipolar cross-correlation rates

  1. Effects of growth and change of food on the δ15N in marine fishes

    Information is limited concerning variation of the δ15N with growth in marine organisms and consequently the effect of growth of marine biota on the δ15N is not yet well understood. The δ15N in 26 species of marine fishes taken from Japanese coastal waters together with 4664 stomach contents of these fishes were examined to investigate the effects of food habits and growth on the δ15N. The mean δ15N for two species that fed mainly on large-size fishes and six species that fed mainly on small-size fishes were 14.5±1.0per mille and 12.8±0.7per mille, respectively. For five species that fed mainly on decapod crustaceans, two species that fed mainly on zooplankton, and three species that fed mainly on benthos (mainly Polychaeta), the δ15N were 13.0±0.7, 9.7±0.9, and 12.2±1.2per mille, respectively. The mean δ15N in the species whose prey were mainly fish or decapod crustaceans was about 3-5per mille higher than the species whose prey was mainly zooplankton. Within the four species that shift their food habits with growth to higher trophic level, the δ15N significantly increased with growth in one species (Pacific cod), while not significant increase in the δ15N with growth in the remaining species. (author)

  2. Nitrogen (15N) recovery from ammonium and nitrate applied to the soil by sugar cane

    An experiment was developed in a field aimed to compare the recovery of the ammonium-15 N and nitrate-15 N by the sugar cane plants harvested mechanically without burning. A rate of 70 kg ha-1 of N was applied as ammonium nitrate, in strip, onto cultural residues. Two lineal meters micropots were used. They received the fertilizer labeled with 15 N. Two treatments were established using labeled ammonium (NH4+-15 N) or nitrate (NO3-15 N). Two months after fertilization, four samples of the aerial part (two lineal meters) for treatment in the portions that did not receive the fertilizer-15 N, were taken in order to evaluated the fitomass production (Mg ha-1) and N-total accumulated (kg ha-1). This evaluation was repeated every two months up to complete five of them. Two leaves (leaves with 3 deg C visible auricle) were collected from plants that were in a middle of the micropots (15 N) and in corresponding positions in the adjacent rows, to evaluated the concentration of 15 N. There was a larger absorption of the nitrate-N (30.5%) than of the ammonium-N (21.2%). On the other hand, in the soil the results showed larger ammonium-15 N residual effect concentration, probably due to microorganism immobilization. (author)

  3. Investigation of the metabolism of colostomized laying hens with 15N-labelled wheat. 5

    In an experiment with 3 colostomized laying hybrids each animal received 80 g pelleted mixed feed and 40 g 15N-labelled wheat with 20.13 atom-% 15N excess (15N') over a period of four days. On the following four days the hens received rations composed in the same way with unlabelled wheat, however in the tissues and organs of the slaughtered hens 15N' was determined in the total N and the amino acids lysine, histidine and arginine in both the segments of the gastro intestinal tract and in its content. The amount of 15N' stomach, small intestine and colon was 43.7%, 27.2% and 29.1%, respectively. The tissue of the small intestine contained, on an average, the highest 15N' in lysine of all the basic amino acids. It was 0.82 atom-% 15N' for lysine, 0.55% for histidine and 0.63% for arginine. The percentage of the 15N' of the basic amino acids from the corresponding total 15N' amount of the charges was 20.5% in the contents of the gastrointestinal tract, 28.0% in the stomach tissue and in the tissues of the small intestine 24.4% of the cecum 21.5% and of the rectum 25.7%. (author)

  4. Utilization of 15N-labelled urea in laying hens. 6

    3 colostomized laying hybrids received a normal ration containing 1% 15N-labelled urea with 96.06% atom-% 15N excess (15N') over six days. Subsequently the same ration with unlabelled urea was given over 2 days, after which the animals were butchered. In the kidneys the 15N' amounted to 1.1 atom-% and 1.8 atom-% in the liver. The TCA soluble N fraction and the ammonia were more highly labelled than the total N. Lysine, histidine and arginine were lowly labelled in the kidneys. This also applies to the liver with the exception of histidine. In the branch-chained and aromatic amino acids of the liver the 15N' was between 0.2 and 0.3 atom-%. The highest labelling of non-essential amino acids was found in glutamic acid with 0.9 atom-% 15N' and aspartic acid with 1.1 atom-% 15 N'. The evaluation of the amino acid in the liver showed that the 6 non-essential amino acids account for two thirds of the total amino acid 15N' whereas the 9 essential ones account for one third of the amino acid 15N' only. (author)

  5. Free backbone carbonyls mediate rhodopsin activation.

    Kimata, Naoki; Pope, Andreyah; Sanchez-Reyes, Omar B; Eilers, Markus; Opefi, Chikwado A; Ziliox, Martine; Reeves, Philip J; Smith, Steven O

    2016-08-01

    Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins. PMID:27376589

  6. A data acquisition backbone core library

    For the new experiments at FAIR new concepts of data acquisition systems have to be developed like the distribution of self-triggered, time stamped data streams over high performance networks for event building. The data acquisition backbone core (DABC) is a general purpose software framework designed for the implementation of such data acquisition systems. It provides the event building over networks like InfiniBand or Gigabit Ethernet. All kinds of data channels (front-end systems) are supported by program plug-ins into functional components of DABC like data input, combiner, scheduler, event builder, analysis and storage components. Commands and parameters of DABC and its application plug-ins are published by DIM servers. A Java based Graphical User Interface provides the dynamic control and visualization of these components. Application specific GUIs can be added. After a testing phase, DABC can be used to develop high performance data acquisition systems. Besides that DABC will be used for the implementation of various test beds needed for the final design of data acquisition systems at FAIR like detector tests, readout components test, and data flow investigations

  7. Factors Controlling the Stable Nitrogen Isotopic Composition (δ15N of Lipids in Marine Animals.

    Elisabeth Svensson

    Full Text Available Lipid extraction of biomass prior to stable isotope analysis is known to cause variable changes in the stable nitrogen isotopic composition (δ15N of residual biomass. However, the underlying factors causing these changes are not yet clear. Here we address this issue by comparing the δ15N of bulk and residual biomass of several marine animal tissues (fish, crab, cockle, oyster, and polychaete, as well as the δ15N of the extracted lipids. As observed previously, lipid extraction led to a variable offset in δ15N of biomass (differences ranging from -2.3 to +1.8 ‰. Importantly, the total lipid extract (TLE was highly depleted in 15N compared to bulk biomass, and also highly variable (differences ranging from -14 to +0.7 ‰. The TLE consisted mainly of phosphatidylcholines, a group of lipids with one nitrogen atom in the headgroup. To elucidate the cause for the 15N-depletion in the TLE, the δ15N of amino acids was determined, including serine because it is one of the main sources of nitrogen to N-containing lipids. Serine δ15N values differed by -7 to +2 ‰ from bulk biomass δ15N, and correlated well with the 15N depletion in TLEs. On average, serine was less depleted (-3‰ than the TLE (-7 ‰, possibly due to fractionation during biosynthesis of N-containing headgroups, or that other nitrogen-containing compounds, such as urea and choline, or recycled nitrogen contribute to the nitrogen isotopic composition of the TLE. The depletion in 15N of the TLE relative to biomass increased with the trophic level of the organisms.

  8. Nitrogen-15 labeled 5S RNA. Identification of uridine base pairs in Escherichia coli 5S RNA by 1H-15N multiple quantum NMR

    Escherichia coli 5S RNA labeled with 15N at N3 of the uridines was isolated from the Sφ-187 uracil auxotroph grown on a minimal medium supplemented with [3-15N]uracil. 1H-15N multiple quantum filtered and 2D chemical shift correlated spectra gave resonances for the uridine imino 1H-15N units whose protons were exchanging slowly with solvent. Peaks with 1H/15N shifts at 11.6/154.8, 11.7/155.0, 11.8/155.5, 12.1/155.0, and 12.2/155.0 ppm were assigned to GU interactions. Two labile high-field AU resonances at 12.6/156.8 and 12.8/157.3 ppm typical of Au pairs in a shielded environment at the end of a helix were seen. Intense AU signals were also found at 13.4/158.5 and 13.6/159.2 ppm where 1H-15N units in normal Watson-Crick pairs resonate. 1H resonances at 10.6 and 13.8 ppm were too weak, presumably because of exchange with water, to give peaks in chemical shift correlated spectra. 1H chemical shifts suggest that the resonance at 13.8 ppm represents a labile AU pair, while the resonance at 10.6 ppm is typical of a tertiary interaction between U and a tightly bound water or a phosphate residue. The NMR data are consistent with proposed secondary structures for 5S RNA

  9. Backbone flexibility in protein design theory and experiment

    Su, Alyce

    The role of backbone flexibility in protein design was studied. First, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein Gβ1 domain (Gβ1). Concerted backbone motion was introduced by varying Gβ1's supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally. Core variants containing as many as six of ten possible mutations retained native-like properties. This result demonstrates that backbone flexibility can be combined with amino acid side-chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of the native parameter values. Second, a general, quantitative design method for computing de novo backbone templates was developed. The method had to compute atomic resolution backbones compatible with the atomistic sequence selection algorithm we were using and it had to be applicable to all protein motifs. We again developed a method that uses super-secondary structure parameters to determine the orientation among secondary structural elements, given a target protein fold. Possible backbone arrangements were screened using a cost function which evaluates core packing, hydrogen bonding, loop closure, and backbone torsional geometry. Given a specified number of residues for each secondary structural element, a family of optimal configurations was found. We chose three motifs to test our method (ββα, βαβ, and αα) since their combination could be used to approximate most possible backbone fold. The best structure found for the ββα motif is similar to a zinc finger, and the best structure for the ββα motif is similar to a segment of a β-barrel. The backbone obtained for the αα motif resembles minimized protein A. Last, our backbone design method was evaluated by testing the thermal stability and structural properties

  10. Identical repeated backbone of the human genome

    Gonzaga-Jauregui Claudia

    2010-01-01

    Full Text Available Abstract Background Identical sequences with a minimal length of about 300 base pairs (bp have been involved in the generation of various meiotic/mitotic genomic rearrangements through non-allelic homologous recombination (NAHR events. Genomic disorders and structural variation, together with gene remodelling processes have been associated with many of these rearrangements. Based on these observations, we identified and integrated all the 100% identical repeats of at least 300 bp in the NCBI version 36.2 human genome reference assembly into non-overlapping regions, thus defining the Identical Repeated Backbone (IRB of the reference human genome. Results The IRB sequences are distributed all over the genome in 66,600 regions, which correspond to ~2% of the total NCBI human genome reference assembly. Important structural and functional elements such as common repeats, segmental duplications, and genes are contained in the IRB. About 80% of the IRB bp overlap with known copy-number variants (CNVs. By analyzing the genes embedded in the IRB, we were able to detect some identical genes not previously included in the Ensembl release 50 annotation of human genes. In addition, we found evidence of IRB gene copy-number polymorphisms in raw sequence reads of two diploid sequenced genomes. Conclusions In general, the IRB offers new insight into the complex organization of the identical repeated sequences of the human genome. It provides an accurate map of potential NAHR sites which could be used in targeting the study of novel CNVs, predicting DNA copy-number variation in newly sequenced genomes, and improve genome annotation.

  11. Enhanced conformational space sampling improves the prediction of chemical shifts in proteins.

    Markwick, Phineus R L; Cervantes, Carla F; Abel, Barrett L; Komives, Elizabeth A; Blackledge, Martin; McCammon, J Andrew

    2010-02-01

    A biased-potential molecular dynamics simulation method, accelerated molecular dynamics (AMD), was combined with the chemical shift prediction algorithm SHIFTX to calculate (1)H(N), (15)N, (13)Calpha, (13)Cbeta, and (13)C' chemical shifts of the ankyrin repeat protein IkappaBalpha (residues 67-206), the primary inhibitor of nuclear factor kappa-B (NF-kappaB). Free-energy-weighted molecular ensembles were generated over a range of acceleration levels, affording systematic enhancement of the conformational space sampling of the protein. We have found that the predicted chemical shifts, particularly for the (15)N, (13)Calpha, and (13)Cbeta nuclei, improve substantially with enhanced conformational space sampling up to an optimal acceleration level. Significant improvement in the predicted chemical shift data coincides with those regions of the protein that exhibit backbone dynamics on longer time scales. Interestingly, the optimal acceleration level for reproduction of the chemical shift data has previously been shown to best reproduce the experimental residual dipolar coupling (RDC) data for this system, as both chemical shift data and RDCs report on an ensemble and time average in the millisecond range. PMID:20063881

  12. Disturbance and topography shape nitrogen availability and δ15N over long-term forest succession

    Forest disturbance and long-term succession can promote open N cycling that increases N loss and soil δ15N values. We examined soil and foliar patterns in N and δ15N, and soil N mineralization, across a topographically complex montane forest landscape influenced by human logging ...

  13. Improvement of differential diagnostics in pseudohermaphroditismus masculinus by means of 15N-labelled amino acids

    In 7 children with male hermaphroditism the N retention test was performed using 15N-glycine (13.5 mg/kg body weight). On testosterone therapy the protein synthesis rate increased significantly while there was a significant decrease in the cumulative 15N excretion

  14. Increased Plant Uptake of Nitrogen from 15N Depleted Fertilizer Using Plant Growth-Promoting Rhizobacteria

    The techniques of 15N isotope have been very useful for determining the behavior and fate of N in soil, including the use efficiency of applied N fertilizers by plants. Our objective in this study was to use 15N isotope techniques to demonstrate that a model plant growth-promoting rhizobacteria (PGP...

  15. Application of 15N amino acid absorption in chronic enteropathy and hepatic diseases in infants

    The aim of this study was to estimate malabsorption status in humans using a 15N stable isotope tracer technique. [15N]-glycine, 98.98 atom %, was synthesized in our institute and was administered orally as a single bolus dose to twelve patients. Six of the 12 subjects studied were healthy and 6 were suspected of having malabsorption. Blood, urine and faecal samples were obtained, proteins in the samples were precipitated with sulphosalicylic acid (5%), the eluate was purified with Dowex 50W-X8 (40mm x 2mm column), and derivatised to form the trifluoroacetyl-butyl esters using standard techniques. Gas chromatographic separation was performed on a glass column 2m X 3mm i.d. packed with EGA 1% on Chromosorb W AW 80-100 mesh. An isotope dilution GC/MS method and Kjeldahl digestion followed by MS analysis of nitrogen gas was performed. 15N isotopomer was used as internal standard. [15N]-Gly elimination in faeces was compared with total 15N elimination in faeces to distinguish artefacts caused by intestinal bacteria. Significant differences in the amount of [15N]-Gly eliminated in urine and faeces between malabsorption and control patients were obtained. It was concluded that more emphasis should be given to the faeces data than to urine because 15N elimination in urine is competitive with 15N incorporation into protein. 12 refs, 4 figs, 4 tabs

  16. Turnover of 15N labelled nitrate with special emphasis on denitrification in the field

    This study establishes a mass balance for 15N-labelled nitrate added to soil planted with a nitrogen-fixing crop (pea) and a non-fixing crop (barley). The results indicate that 15N unaccounted for in a mass balance is not necessarily lost by denitrification. Processes such as volatilization of ammonia should also be considered. 1 fig

  17. Synthesis and isotope-ratio analysis of methyl nitrite-15N

    Methyl nitrite-15N was synthesised on a 0.1 mole scale by the esterification of methanol by aqueous H15NO2. The method is simple and efficient, and provides analytically pure CH3O15NO. A method for determining the 15N enrichment of CH3O15NO is described. (author)

  18. Utilization of 15N-labelled urea in laying hens. 8

    3 colostomized laying hybrids received orally with a conventional ration 1% urea with 96.06 atom-% 15N excess (15N') over a period of 6 days. In the period of the experiment every hen consumed 2.87 g 15N'. After another 2 days, on which they received conventional feed urea, the animals were butchered. 15N' was determined in the total N and in 15 amino acids of the oviduct. Of the 15 amino acids the labelling of glutamic acid, glycine and serine was highest and on average amounted to 0.80, 0.66 and 0.67 atom-% 15N', resp. In lysine and arginine only 0.10 and 0.11 atom-% 15N' could be detected. The amino acid N with natural isotopic frequency amounted to a quarter for the basic amino acids, a tenth for the branched chain ones and for the non-essential ones (glutamic acid, aspartic acid, serine, glycine, alanine, proline) a third of the total oviduct 14N. The average quota of 15N' is only 3.6%, that of the branched chain amino acids 4.5 and that of the non-essential ones 21.1%. Consequently, the 15N' of the urea is mainly used for the synthesis of the non-essential amino acids of the oviduct. (author)

  19. Utilization of 15N-labelled urea in laying hens. 2

    In an N metabolism experiment 3 colostomized laying hybrids received 2870 mg 15N excess (15N') per animal in 6 days in the form of urea with their conventional feed rations. During the 8-day experiment the 21 eggs laid were separated into egg-shell, white of egg and yolk. Weight, N content and 15N' of the individual fractions of the eggs were determined. On an average 4.6% of the heavy nitrogen was in the egg-shells, 50% in the white of egg and 45.5% in the yolk. 2.8%, 4.5% and 5.5% (hens 1 - 3) of the 15N' consumed were detected in the eggs. The maximum 15N' output in the white of egg was reached on the 6th day, whereas 15N' output in the yolk showed a nearly linear increase in the time of the experiment. The results show that labelled nitrogen from urea is incorporated into the egg to a lower degree than after the feeding of 15N-labelled proteins and that the development of its incorporation into the white of egg and the yolk differ from that after the feeding of 15N-labelled native proteins. (author)

  20. Global Fold of Human Cannabinoid Type 2 Receptor Probed by Solid-State 13C-, 15N-MAS NMR and Molecular Dynamics Simulations

    Kimura, Tomohiro; Vukoti, Krishna; Lynch, Diane L.; Hurst, Dow P.; Grossfield, Alan; Pitman, Michael C.; Reggio, Patricia H.; Yeliseev, Alexei A.; Gawrisch, Klaus

    2013-01-01

    The global fold of human cannabinoid type 2 (CB2) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state 13C- and 15N magic-angle spinning (MAS) NMR, in combination with chemical-shift prediction from a structural model of the receptor obtained by microsecond-long molecular dynamics (MD) simulations. Uniformly 13C-, and 15N-labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into l...

  1. Efficient Algorithms for Extension of Mobile Backbone Networks

    Eswaramoorthi. R

    2012-03-01

    Full Text Available Network wide communication is an essential criterion for all wireless sensor networks, to transmit the collected data from environment to base station (sink node in an efficient way. The wide network coverage is provided by constructing Mobile Backbone Networks (MBN, which are dynamic networks. These networks have two types of nodes. They are Mobile backbone nodes and Regular nodes. The Mobile backbone nodes have superior mobility and communication capability than regular nodes. All the information needs to be routed through mobile backbone nodes to regular nodes. The communication between clusters is done through backbone node so that transmission overhead is less. In this paper, we are mainly concentrates on throughput optimization and assigning new regular nodes. First the throughput range is calculated for each cluster, and then data packets are transmitted in such a way that the calculated range of throughput for each cluster is satisfied. The number of regular nodes that can be successfully assigned to mobile backbone nodes can be improved by means of adopting network design formulation technique. In case of any failure of mobile backbone node ,new cluster head is elected based on high energy first (HEF algorithm, where the residual energy of the nodes are considered for election.

  2. Radiation safety system (RSS) backbones: Design, engineering, fabrication and installation

    The Radiation Safety System (RSS) Backbones are part of an electrical/electronic/mechanical system insuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS Backbones control the safety fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low energy beam transport. The Backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the Backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two Linac Backbone segments and experimental area segments form a continuous cable plant over 3,500 feet from beam plugs to the tip on the longest tail. The Backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely

  3. Radiation safety system (RSS) backbones: Design, engineering, fabrication and installation

    Wilmarth, J.E.; Sturrock, J.C.; Gallegos, F.R.

    1998-12-01

    The Radiation Safety System (RSS) Backbones are part of an electrical/electronic/mechanical system insuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS Backbones control the safety fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low energy beam transport. The Backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the Backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two Linac Backbone segments and experimental area segments form a continuous cable plant over 3,500 feet from beam plugs to the tip on the longest tail. The Backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely.

  4. Syntheses of 15N-labeled pre-queuosine nucleobase derivatives

    Jasmin Levic

    2014-08-01

    Full Text Available Pre-queuosine or queuine (preQ1 is a guanine derivative that is involved in the biosynthetic pathway of the hypermodified tRNA nucleoside queuosine (Que. The core structure of preQ1 is represented by 7-(aminomethyl-7-deazaguanine (preQ1 base. Here, we report the synthesis of three preQ1 base derivatives with complementary 15N-labeling patterns, utilizing [15N]-KCN, [15N]-phthalimide, and [15N3]-guanidine as cost-affordable 15N sources. Such derivatives are required to explore the binding process of the preQ1 base to RNA targets using advanced NMR spectroscopic methods. PreQ1 base specifically binds to bacterial mRNA domains and thereby regulates genes that are required for queuosine biosynthesis.

  5. Utilization of fertilizer and stored nitrogen by asparagus and kiwifruit estimated using 15N

    The efficiency of recovery of 15N enriched nitrogen fertilizer was examined in field trials on two contrasting mature perennial crops: a vegetable (asparagus) and a woody deciduous vine (kiwifruit). In the asparagus experiment, 50 kg N/ha were applied either prior to fern growth (early summer) or pre-harvest (early spring). In the former treatment, 15N uptake was rapid during the period of fern growth (summer and autumn) and by early winter most 15N had been stored in crown and root material. In contrast, uptake of 15N applied pre-harvest was slow, indicating that most of the N for spear production was from remobilization of stored N. Removal of added 15N in produce over two harvest years was small. 8 refs, 3 tabs

  6. Standardized 15N tracer methods for the evaluation of the plasma protein turnover in clinical practice. 1

    Methods for quantitative isolation of plasma proteins or groups of proteins (total plasma or serum proteins, fibrin, total globulines, α, β, γ-globolines, albumin) are described based on combination of chromatography with precipitation and extraction techniques. These methods are adapted to the special requirements of 15N analysis. They can be performed in clinic-chemical standard laboratories without special apparatuses or devices. The described procedures are the biochemico-analytical basis for the quantitative evaluation of tracer kinetics data by means of mathematic modelling. (author)

  7. Using a macroalgal δ15N bioassay to detect cruise ship waste water effluent inputs

    Highlights: → Green macroalgae exposed to nutrient solutions exhibited changes in tissue 15N signatures. → Macroalgae exhibited no fractionation with NO3 and slight fractionation with NH4. → Algae exposed to cruise ship waste water had increased tissue δ15N indicating a heavy N source. → Field bioassays exhibited decreased δ15N indicating isotopically light riverine δ15N-NO3 was likely the dominant N source. → Algal bioassays could not detect a δ15N cruise ship waste water signal in this system. - Abstract: Green macroalgae bioassays were used to determine if the δ15N signature of cruise ship waste water effluent (CSWWE) could be detected in a small harbor. Opportunistic green macroalgae (Ulva spp.) were collected, cultured under nutrient depleted conditions and characterized with regard to N content and δ15N. Samples of algae were used in controlled incubations to evaluate the direction of isotope shift from exposure to CSWWE. Algae samples exposed to CSWWE exhibited an increase of 1-2.5 per mille in δ15N values indicating that the CSWWE had an enriched isotope signature. In contrast, algae samples exposed to field conditions exhibited a significant decrease in the observed δ15N indicating that a light N source was used. Isotopically light, riverine nitrogen derived from N2-fixing trees in the watershed may be a N source utilized by algae. These experiments indicate that the δ15N CSWWE signature was not detectable under the CSWWE loading conditions of this experiment.

  8. Utilization of 15N-labelled urea in laying hens. 9

    For studying the incorporation of the 15N labelled urea into individual organs and tissues 3 colostomized laying hens were butchered after they had received 1% urea (96.06 atom-% 15N excess) with a high quality ration over a period of six days and after receiving conventional urea for another two days. Nitrogen and atom-% 15N excess (15N') were determined in the bones, the feathers and the remaining body (skin, lungs and windpipe, head with comb and wattle, lower leg without bones and with skin, pancreas and fatty tissue). In the remaining body the atom-% 15N' was determined in 15 amino acids. The labelling in the remaining body and the bones was approximately the same and averaged 0.37 atom-% 15N'. A significantly lower relative frequency could be detected in the feathers. The lysine of the remaining body contained only 0.04 atom-% 15N', tyrosine 0.06, histidine and arginine 0.07. The phenylalanine and proline molecules were labelled with 0.11 atom-% 15N'. Most 15N' was incorporated in serine and glutamic acid with over 0.30 atom-%. In the six non-essential amino acids out of the 15 amino acids studied, 48.6 of the non-isotopic nitrogen of the total N of the remaining body and 70.7% of the isotopic nitrogen of total 15N' could be detected. Consequently the urea N is mainly used for the synthesis of the non-essential amino acids, with its utilization being very low. (author)

  9. Security in Packet-Switched Land Mobile Radio Backbone Networks

    Thomschutz, Hans Olaf Rutger

    2005-01-01

    Spurred by change in government regulations and to leverage lower-cost technology and services, many land mobile radio (LMR) operators have begun transitioning from circuit-switched to packet-switched backbone networks to handle their future communication needs. Due to the unique demands of packet-switched backbone networks for LMR, it may not be wise to carry over the previously implemented security methods used with circuit-switch systems or to treat an LMR backbone as a regular packet-swit...

  10. Labelling of sewage with 13C and 15N isotopes

    Treatment of sewage water varies with the type and level of technology applied. As a result, sewage sludges vary in composition. In Western Europe, a combination of mechanical, biological, and chemical treatments is commonly applied. The biological treatment of sewage water - the activated sludge process - results in removal of carbon and nitrogen through immobilization in microbes. With strong aeration of the wastewater, energy-rich substrates and nutrients are assimilated by aerobic microbes and a large microbial biomass results. The biomass consists mainly of living microbial cells and components of dying and dead cells, but also of colloidal particles and metal ions bound to the surfaces of the microbes. The organic matter produced during aeration - the biological sludge - is removed by settlement. The biological treatment of wastewater was the starting point for the labelling procedure of sewage sludge. Labelling of waste products with stable tracer isotopes can be done in two ways: (i) labelling of the original material from which wastes are generated, e.g. by labelling of the diet fed to animals; and (ii) labelling during the biological turnover through addition of nitrogen or carbon compounds to wastes. In this study, tracers were added to wastewater during biological treatment

  11. NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23-230)

    A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15N, 13C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15N relaxation rates of unfolded polypeptides in high resolution constant-time [1H, 15N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence

  12. Detection of (15)NNH+ in L1544: non-LTE modelling of dyazenilium hyperfine line emission and accurate (14)N/(15)N values

    Bizzocchi, Luca; Leonardo, Elvira; Dore, Luca

    2013-01-01

    Samples of pristine Solar System material found in meteorites and interplanetary dust particles are highly enriched in (15)N. Conspicuous nitrogen isotopic anomalies have also been measured in comets, and the (14)N/(15)N abundance ratio of the Earth is itself larger than the recognised pre-solar value by almost a factor of two. Ion--molecules, low-temperature chemical reactions in the proto-solar nebula have been repeatedly indicated as responsible for these (15)N-enhancements. We have searched for (15)N variants of the N2H+ ion in L1544, a prototypical starless cloud core which is one of the best candidate sources for detection owing to its low central core temperature and high CO depletion. The goal is the evaluation of accurate and reliable (14)N/(15)N ratio values for this species in the interstellar gas. A deep integration of the (15)NNH+ (1-0) line at 90.4 GHz has been obtained with the IRAM 30 m telescope. Non-LTE radiative transfer modelling has been performed on the J=1-0 emissions of the parent and ...

  13. Variable δ15N Diet-Tissue Discrimination Factors among Sharks: Implications for Trophic Position, Diet and Food Web Models

    Olin, Jill A.; Hussey, Nigel E.; Alice Grgicak-Mannion; Mark W Fritts; Wintner, Sabine P.; Fisk, Aaron T.

    2013-01-01

    The application of stable isotopes to characterize the complexities of a species foraging behavior and trophic relationships is dependent on assumptions of δ(15)N diet-tissue discrimination factors (∆(15)N). As ∆(15)N values have been experimentally shown to vary amongst consumers, tissues and diet composition, resolving appropriate species-specific ∆(15)N values can be complex. Given the logistical and ethical challenges of controlled feeding experiments for determining ∆(15)N values for lar...

  14. NMR characterization of structure, backbone dynamics, and glutathione binding of the human macrophage migration inhibitory factor (MIF).

    Mühlhahn, P; Bernhagen, J; Czisch, M; Georgescu, J; Renner, C; Ross, A; Bucala, R; Holak, T A

    1996-10-01

    Human macrophage migration inhibitory factor is a 114 amino acid protein that belongs to the family of immunologic cytokines. Assignments of 1H, 15N, and 13C resonances have enabled the determination of the secondary structure of the protein, which consists of two alpha-helices (residues 18-31 and 89-72) and a central four-stranded beta-sheet. In the beta-sheet, two parallel beta-sheets are connected in an antiparallel sense. From the total of three cysteines present in the primary structure of MIF, none was found to form disulfide bridges. 1H-15N heteronuclear T1, T2, and steady-state NOE measurements indicate that the backbone of MIF exists in a rigid structure of limited conformational flexibility (on the nanosecond to picosecond time scale). Several residues located in the loop regions and at the N termini of two helices exhibit internal motions on the 1-3 ns time scale. The capacity to bind glutathione was investigated by titration of a uniform 15N-labeled sample and led us to conclude that MIF has, at best, very low affinity for glutathione. PMID:8897610

  15. Bromine recovery in residual solutions generated in the 15 N isotopic determination methodology (Rittenberg, 1946)

    The isotopic determination of 15 N (Rittenberg, 1946) is a methodology used in the Laboratory of Isotope Stable (CENA/USP). In this procedure, in the oxidation of nitrogen species for N2, solution of Li Br O is used, generating as residue 50 L y-1 of solution contends Li Br and Li Br O. Seeking to recover the bromine contained in that residue, very toxic substance, a special line was built composed by reaction balloons (1 and 2 liters), addition funnel, gas flow regulator and connections in glass. In the system proposed, after the acidification (sulfuric acid) of the alkaline residual solution, the liberated bromine (Br2) it was then dragged by flow of nitrogen and reacted with solution of LiOH. That reaction facilitated the production of Li Br O in solution (Efficiency = 82±2%), that was reused later on same analytic procedure. The high cost of the liquid bromine is another attractiveness that corroborates the employment of the developed procedure. They took place isotopic determinations using the recovered solutions and prepared, and the observed values didn't show statistical difference (T test of Student). The presented procedure is part of the Management Program of Chemical Residues of CENA/USP, which seeks to destine the residues of responsibility of the institution appropriately, forming professionals to the practices of environmental management. (author)

  16. Nitrogen fertilizer (15N leaching in a central pivot fertigated coffee crop

    Rafael Pivotto Bortolotto

    2012-08-01

    Full Text Available Nitrogen has a complex dynamics in the soil-plant-atmosphere system. N fertilizers are subject to chemical and microbial transformations in soils that can result in significant losses. Considering the cost of fertilizers, the adoption of good management practices like fertigation could improve the N use efficiency by crops. Water balances (WB were applied to evaluate fertilizer N leaching using 15N labeled urea in west Bahia, Brazil. Three scenarios (2008/2009 were established: i rainfall + irrigation the full year, ii rainfall only; and iii rainfall + irrigation only in the dry season. The water excess was considered equal to the deep drainage for the very flat area (runoff = 0 with a water table located several meters below soil surface (capillary rise = 0. The control volume for water balance calculations was the 0 - 1 m soil layer, considering that it involves the active root system. The water drained below 1 m was used to estimate fertilizer N leaching losses. WB calculations used the mathematic model of Penman-Monteith for evapotranspiration, considering the crop coefficient equal to unity. The high N application rate associated to the high rainfall plus irrigation was found to be the main cause for leaching, which values were 14.7 and 104.5 kg ha-1 for the rates 400 and 800 kg ha-1 of N, corresponding to 3.7 and 13.1 % of the applied fertilizer, respectively.

  17. A sampling approach for protein backbone fragment conformations.

    Yu, J Y; Zhang, W

    2013-01-01

    In protein structure prediction, backbone fragment bias information can narrow down the conformational space of the whole polypeptide chain significantly. Unlike existing methods that use fragments as building blocks, the paper presents a probabilistic sampling approach for protein backbone torsion angles by modelling angular correlation of (phi, psi) with a directional statistics distribution. Given a protein sequence and secondary structure information, this method samples backbone fragments conformations by using a backtrack sampling algorithm for the hidden Markov model with multiple inputs and a single output. The proposed approach is applied to a fragment library, and some well-known structural motifs are sampled very well on the optimal path. Computational results show that the method can help to obtain native-like backbone fragments conformations. PMID:23777175

  18. LOAD AWARE ADAPTIVE BACKBONE SYNTHESIS IN WIRELESS MESH NETWORKS

    Yuan Yuan; Zheng Baoyu

    2009-01-01

    Wireless Mesh Networks (WMNs) are envisioned to support the wired backbone with a wireless Backbone Networks (BNet) for providing internet connectivity to large-scale areas.With a wide range of internet-oriented applications with different Quality of Service (QoS) requirement,the large-scale WMNs should have good scalability and large bandwidth.In this paper,a Load Aware Adaptive Backbone Synthesis (LAABS) algorithm is proposed to automatically balance the traffic flow in the WMNs.The BNet will dynamically split into smaller size or merge into bigger one according to statistic load information of Backbone Nodes (BNs).Simulation results show LAABS generates moderate BNet size and converges quickly,thus providing scalable and stable BNet to facilitate traffic flow.

  19. δ15N of seagrass leaves for monitoring anthropogenic nutrient increases in coral reef ecosystems

    In a coral reef environment, a slight increase in dissolved inorganic nitrogen (DIN;≥1.0 μM) can alter the ecosystem via macroalgal blooms. We collected seagrass leaves from the tropical and subtropical Pacific Ocean in five countries and examined the interactions between nutrient concentrations (C, N, P), molar ratios of nutrients, and δ15N to find a possible indicator of the DIN conditions. Within most sites, the concentrations of nutrients and their molar ratios showed large variations owing to species-specific values. On the other hand, almost identical δ15N values were found in seagrass leaves of several species at each site. The correlations between δ15N and nutrient concentrations and between δ15N and molar ratios of nutrients suggested that nutrient availability did not affect the δ15N value of seagrass leaves by altering the physiological condition of the plants. Increases in δ15N of seagrass leaves mostly matched increases in DIN concentrations in the bottom water. We suggest that δ15N in seagrass leaves can be a good tool to monitor time-integrated decrease/increase of DIN concentrations at a site, both in the water column and the interstitial water

  20. 15N-labeled nitrogen from green manure and ammonium sulfate utilization by the sugarcane ratoon

    Legumes as green manure are alternative sources of nitrogen (N) for crops and can supplement or even replace mineral nitrogen fertilization due to their potential for biological nitrogen fixation (BNF). The utilization of nitrogen by sugarcane (Saccharum spp.) fertilized with sunn hemp (Crotalaria juncea L.) and ammonium sulfate (AS) was evaluated using the 15N tracer technique. N was added at the rate of 196 and 70 kg ha-1 as 15N-labeled sunn hemp green manure (SH) and as ammonium sulfate (AS), respectively. Treatments were: (I) Control; (II) AS15N; (III) SH15N + AS; (IV) SH15N; and (V) AS15N + SH. Sugarcane was cultivated for five years and was harvested three times. 15N recovery was evaluated in the two first harvests. In the sum of the three harvests, the highest stalk yields were obtained with a combination of green manure and inorganic N fertilizer; however, in the second cutting the yields were higher where SH was used than in plots with AS. The recovery of N by the first two consecutive harvests accounted for 19 to 21% of the N applied as leguminous green manure and 46 to 49% of the N applied as AS. The amounts of inorganic N, derived from both N sources, present in the 0-0.4 m layer of soil in the first season after N application and were below 1 kg ha-1. (author)

  1. Determination of nitrogenase activity of induced cucumber nodules by 15N Trace method

    The author reports the determination results of nitrogenase activity of induced cucumber root nodules by 15N trace method. The root systems bearing induced nodules of cucumber were exposed to a gas mixture containing 15N2 for 48 h and partial root systems soaked in free-nitrogen culture solution simultaneously. After exposure the 15N content in the modulated root systems of cucumber is 0.431 Atom % 15N by mass spectrometric analysis, whereas in the contrast samples without exposure to 15N is 0.369 Atom % 15N. The statistical t test for the results of 15N trace experiments is: t = 3.15 > t0.01 = 2.819. It has been demonstrated that the nitrogenase activity in cucumber nodules is at a remarkable level of 99.9%. The nitrogenase activity in detached nodules of cucumber was also determined by conventional acetylene reduction method. In both methods clear evidences of nitrogenase activity were obtained for the induced nodules of cucumber

  2. Alkaline Hydrolysis/Polymerization of 2,4,6-Trinitrotoluene: Characterization of Products by 13C and 15N NMR

    Thorn, K.A.; Thorne, P.G.; Cox, L.G.

    2004-01-01

    Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and 15N-labeled TNT were analyzed by elemental analysis and 13C and 15N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp 3- and 86% sp2-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp3- and 67% sp 2-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The 15N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to

  3. 使用Backbone JS搭建SPA

    吕婷

    2012-01-01

    Backbone JSR从2010年发布以来,受到了业界的广泛关注。“豆瓣说”和“豆瓣阅读(阅读器)”是两款以它为基础框架搭建的SPA,本文将结合这两款产品,向读者介绍BackboneJS的方方面面。

  4. Side chain and backbone ordering in a polypeptide

    Wei, Y; Hansmann, U H E

    2006-01-01

    We report results from multicanonical simulations of polyglutamic acid chains of length of ten residues. For this simple polypeptide we observe a decoupling of backbone and side-chain ordering in the folding process. While the details of the two transitions vary between the peptide in gas phase and in an implicit solvent, our results indicate that, independent of the specific surroundings, upon continuously lowering the temperature side-chain ordering occurs only after the backbone topology is completely formed.

  5. Sequence-specific {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments for intestinal fatty-acid-binding protein complexed with palmitate (15.4 kDA)

    Hodsdon, M.E.; Toner, J.J.; Cistola, D.P. [Washington Univ. School of Medicine, St. Louis, MO (United States)

    1994-12-01

    Intestinal fatty-acid-binding protein (I-FABP) belongs to a family of soluble, cytoplasmic proteins that are thought to function in the intracellular transport and trafficking of polar lipids. Individual members of this protein family have distinct specificities and affinities for fatty acids, cholesterol, bile salts, and retinoids. We are comparing several retinol- and fatty-acid-binding proteins from intestine in order to define the factors that control molecular recognition in this family of proteins. We have established sequential resonance assignments for uniformly {sup 13}C/{sup 15}N-enriched I-FABP complexed with perdeuterated palmitate at pH7.2 and 37{degrees}C. The assignment strategy was similar to that introduced for calmodulin. We employed seven three-dimensional NMR experiments to establish scalar couplings between backbone and sidechain atoms. Backbone atoms were correlated using triple-resonance HNCO, HNCA, TOCSY-HMQC, HCACO, and HCA(CO)N experiments. Sidechain atoms were correlated using CC-TOCSY, HCCH-TOCSY, and TOCSY-HMQC. The correlations of peaks between three-dimensional spectra were established in a computer-assisted manner using NMR COMPASS (Molecular Simulations, Inc.) Using this approach, {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments have been established for 120 of the 131 residues of I-FABP. For 18 residues, amide {sup 1}H and {sup 15}N resonances were unobservable, apparently because of the rapid exchange of amide protons with bulk water at pH 7.2. The missing amide protons correspond to distinct amino acid patterns in the protein sequence, which will be discussed. During the assignment process, several sources of ambiguity in spin correlations were observed. To overcome this ambiguity, the additional inter-residue correlations often observed in the HNCA experiment were used as cross-checks for the sequential backbone assignments.

  6. Light-mediated 15N fractionation in Caribbean gorgonian octocorals: implications for pollution monitoring

    Baker, D. M.; Kim, K.; Andras, J. P.; Sparks, J. P.

    2011-09-01

    The stable nitrogen isotope ratio ( δ 15N) of coral tissue is a useful recorder of anthropogenic pollution in tropical marine ecosystems. However, little is known of the natural environmentally induced fractionations that affect our interpretation of coral δ 15N values. In symbiotic scleractinians, light affects metabolic fractionation of N during photosynthesis, which may confound the identification of N pollution between sites of varied depth or turbidity. Given the superiority of octocorals for δ 15N studies, our goal was to quantify the effect of light on gorgonian δ 15N in the context of monitoring N pollution sources. Using field collections, we show that δ 15N declined by 1.4‰ over 20 m depth in two species of gorgonians, the common sea fan, Gorgonia ventalina, and the slimy sea plume, Pseudopterogorgia americana. An 8-week laboratory experiment with P. americana showed that light, not temperature causes this variation, whereby the lowest fractionation of the N source was observed in the highest light treatment. Finally, we used a yearlong reciprocal depth transplant experiment to quantify the time frame over which δ 15N changes in G. ventalina as a function of light regime . Over the year, δ 15N was unchanged and increased slightly in the deep control colonies and shallow colonies transplanted to the deep site, respectively. Within 6 months, colonies transplanted from deep to shallow became enriched by 0.8‰, mirroring the enrichment observed in the shallow controls, which was likely due to the combined effect of an increase in the source δ 15N and reduced fractionation. We conclude that light affects gorgonian δ 15N fractionation and should be considered in sampling designs for N pollution monitoring. However, these fractionations are small relative to differences observed between natural and anthropogenic N sources.

  7. Utilization of 15N-Diammonium Phosphate by Ruminants to Produce Milk and Meat Proteins

    The authors investigated the alimentary role of diammonium phosphate (DAP) in ruminants. For this study DAP labelled with 15N was used; analysis of the 15N atomic per cent excess was made with an Italelettronica mass spectrophotometer (model SP 21 F) and the amino acid determination by a Beckman-Spinco amino acid analyser (model 120B) fitted with a preparative column. For the experiment 7 g of DAP at 15 and 20 at. % excess 15N were administered once to mature lactating and non-lactating sheep, respectively. The measurement of 15N in the protein and isolated amino acids of milk and meat showed: (1) The milk protein produced in the first 24 h contained the highest atomic per cent excess of 15SN, 0.093; (2) That the supplemental 15N was found in all the amino acids of milk proteins except tryptophane. The atomic per cent excess of 15N was observed to vary between the various amino acids. These results confirmed previous observations on bacterial protein synthesized from DAP. (3) Muscle protein 15N maximized on the third day after administration of the 15N-DAP, with an atomic per cent excess of 0.040; (4) The atomic per cent excess of 15N in the individual amino acids of muscle protein is significant in only two amino' acids, serine and cystine; and (5) That after 8 d of adaptation there are no traces of DAP in milk or meat proteins, urine or faeces. The authors conclude that the ruminant, after a period of adaptation and through the mediation of ruminant microorganisms, is able to use the nitrogen of diammonium phosphate for the synthesis of milk and meat proteins. (author)

  8. Absorption and metabolization of orally administered D-[α-15N]lysine and L-[α-15N]lysine with regard to the metabolism of intestinal bacteria

    Absorption of D-[α-15N]lysine and L-[α-15N]lysine following oral single pulse-labelling at a dosage of 5 mg 15N'/kg body weight was compared in four subjects aged 4 to 14 months. The wastages of 15N' in the feces ranged from 0.3 to 5% of the input implying comparably high absorption rates of both the lysine enantiomers. Only about 7.6% of the 15N from the α-amino groups were found in the urine after loading with L-[α-15N]lysine. In contrast, about 80.2% of the 15N' dose from D-[α-15N]lysine were eliminated renally. However, 18.5% of the 15N' dose on an average were retained after D-[α-15N]lysine administration. This is certainly due to a partial desamination of D-lysine. The fecal bacteria isolated from the feces contained no or only small amounts of 15N' after D-[α-15N]lysine loading. Following L-[α-15N]lysine administration a measurable 15N enrichment of the fecal bacteria of up to 0.09 at.% excess was achieved in almost all cases. (author)

  9. Large-scale measurement and modeling of backbone Internet traffic

    Roughan, Matthew; Gottlieb, Joel

    2002-07-01

    There is a brewing controversy in the traffic modeling community concerning how to model backbone traffic. The fundamental work on self-similarity in data traffic appears to be contradicted by recent findings that suggest that backbone traffic is smooth. The traffic analysis work to date has focused on high-quality but limited-scope packet trace measurements; this limits its applicability to high-speed backbone traffic. This paper uses more than one year's worth of SNMP traffic data covering an entire Tier 1 ISP backbone to address the question of how backbone network traffic should be modeled. Although the limitations of SNMP measurements do not permit us to comment on the fine timescale behavior of the traffic, careful analysis of the data suggests that irrespective of the variation at fine timescales, we can construct a simple traffic model that captures key features of the observed traffic. Furthermore, the model's parameters are measurable using existing network infrastructure, making this model practical in a present-day operational network. In addition to its practicality, the model verifies basic statistical multiplexing results, and thus sheds deep insight into how smooth backbone traffic really is.

  10. Radiative p 15N Capture in the Region of Astrophysical Energies

    Dubovichenko, S. B.; Burtebaev, N.; Dzhazairov-Kakhramanov, A. V.; Alimov, D. K.

    2016-06-01

    Within the framework of the modified potential cluster model with classification of orbital states according to the Young schemes, the possibility of describing experimental data for the astrophysical S-factor of p 15N radiative capture at energies from 50 to 1500 keV is considered. It is shown that on the basis of M1 and E1 transitions from various p 15N scattering states to the ground state of the 16O nucleus in the p 15N channel it is entirely possible to successfully explain the overall behavior of the S-factor in the considered energy region in the presence of two resonances.