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Sample records for 15n resonance assignments

  1. 1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein.

    Buchko, Garry W.; Bekhazi, Jacky G.; Cort, John R.; Valentine, Nancy B.; Snead, Malcolm L.; Shaw, Wendy J.

    2008-06-01

    Amelogenin is the predominant matrix protein in developing dental enamel. Making extensive use of residue-specific 15N-labeled amino acids samples, the majority of the main and side chain resonances for murine amelogenin were assigned in 2% aqueous acetic acid at pH 3.0. This research was performed at Pacific Northwest National Laboratory, operated by Battelle for the US-DOE. A large part of this research was performed at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by U.S. Department of Energy’s Office of Biological and Environmental Research (BER) program located at Pacific Northwest National Laboratory (PNNL).

  2. 13C, 15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form

    The partial 15N and 13C solid-state NMR resonance assignment of the HET-s prion protein fragment 218-289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20-40 kHz using exclusively adiabatic polarization-transfer schemes. The resonance assignment within each residue is based on two-dimensional 13C--13C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional 15N--13C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad resonance lines that escaped detection in the experiments applied. The line widths of the observed resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues

  3. 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 ...

  4. 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

  5. Towards fully automated structure-based NMR resonance assignment of 15N-labeled proteins from automatically picked peaks

    Jang, Richard

    2011-03-01

    In NMR resonance assignment, an indispensable step in NMR protein studies, manually processed peaks from both N-labeled and C-labeled spectra are typically used as inputs. However, the use of homologous structures can allow one to use only N-labeled NMR data and avoid the added expense of using C-labeled data. We propose a novel integer programming framework for structure-based backbone resonance assignment using N-labeled data. The core consists of a pair of integer programming models: one for spin system forming and amino acid typing, and the other for backbone resonance assignment. The goal is to perform the assignment directly from spectra without any manual intervention via automatically picked peaks, which are much noisier than manually picked peaks, so methods must be error-tolerant. In the case of semi-automated/manually processed peak data, we compare our system with the Xiong-Pandurangan-Bailey- Kellogg\\'s contact replacement (CR) method, which is the most error-tolerant method for structure-based resonance assignment. Our system, on average, reduces the error rate of the CR method by five folds on their data set. In addition, by using an iterative algorithm, our system has the added capability of using the NOESY data to correct assignment errors due to errors in predicting the amino acid and secondary structure type of each spin system. On a publicly available data set for human ubiquitin, where the typing accuracy is 83%, we achieve 91% accuracy, compared to the 59% accuracy obtained without correcting for such errors. In the case of automatically picked peaks, using assignment information from yeast ubiquitin, we achieve a fully automatic assignment with 97% accuracy. To our knowledge, this is the first system that can achieve fully automatic structure-based assignment directly from spectra. This has implications in NMR protein mutant studies, where the assignment step is repeated for each mutant. © Copyright 2011, Mary Ann Liebert, Inc.

  6. 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.

  7. 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

  8. 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.

  9. Near-complete (1)H, (13)C, (15)N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T.

    Kulminskaya, Natalia V; Yoshimura, Yuichi; Runager, Kasper; Sørensen, Charlotte S; Bjerring, Morten; Andreasen, Maria; Otzen, Daniel E; Enghild, Jan J; Nielsen, Niels Chr; Mulder, Frans A A

    2016-04-01

    The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D (1)H-(15)N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain (1)H, (13)C and (15)N resonances for unfolded FAS1-4 A546T at 25 °C. PMID:26275916

  10. 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

  11. 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.

  12. (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

  13. 1H, 13C, and 15N resonance assignment of the N-terminal domainof Mason-Pfizer monkey virus capsid protein, CA 1-140

    Macek, Pavel; Žídek, L.; Rumlová, Michaela; Pichová, Iva; Sklenář, V.

    2008-01-01

    Roč. 2, č. 1 (2008), s. 43-45. ISSN 1874-2718 R&D Projects: GA MŠk LC545; GA MŠk(CZ) LC06030; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z40550506 Keywords : nmr * assignment * capsid protein Subject RIV: EE - Microbiology, Virology Impact factor: 0.015, year: 2008

  14. Assignment of 1HN, 15N, 13Cα, 13CO and 13Cβ resonances in a 67 kDa p53 dimer using 4D-TROSY NMR spectroscopy

    The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1HN, 15N, 13Cα, 13CO and 13Cβ resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA

  15. 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

  16. 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.

  17. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional 15N-1H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.

  18. 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.

  19. 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.

  20. (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.

  1. 1H, 13C and 15N NMR assignments of phenazopyridine derivatives.

    Burgueño-Tapia, Eleuterio; Mora-Pérez, Yolanda; Morales-Ríos, Martha S; Joseph-Nathan, Pedro

    2005-03-01

    Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional (1)H, (13)C and (15)N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the (15)N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry. PMID:15625718

  2. Complete (1)H, (15)N and (13)C assignment of trappin-2 and (1)H assignment of its two domains, elafin and cementoin.

    Loth, Karine; Alami, Soha Abou Ibrahim; Habès, Chahrazed; Garrido, Solène; Aucagne, Vincent; Delmas, Agnès F; Moreau, Thierry; Zani, Marie-Louise; Landon, Céline

    2016-04-01

    Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete (1)H, (15)N and (13)C resonance assignment of the recombinant trappin-2 and the (1)H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2. PMID:26878852

  3. MUSIC in Triple-Resonance Experiments: Amino Acid Type-Selective 1H- 15N Correlations

    Schubert, Mario; Smalla, Maika; Schmieder, Peter; Oschkinat, Hartmut

    1999-11-01

    Amino acid type-selective triple-resonance experiments can be of great help for the assignment of protein spectra, since they help to remove ambiguities in either manual or automated assignment procedures. Here, modified triple-resonance experiments that yield amino acid type-selective 1H-15N correlations are presented. They are based on novel coherence transfer schemes, the MUSIC pulse sequence elements, that replace the initial INEPT transfer and are selective for XH2 or XH3 (X can be 15N or 13C). The desired amino acid type is thereby selected based on the topology of the side chain. Experiments for Gly (G-HSQC); Ala (A-HSQC); Thr, Val, Ile, and Ala (TAVI-HSQC); Thr and Ala (TA-HSQC), as well as Asn and Gln (N-HSQC and QN-HSQC), are described. The new experiments are recorded as two-dimensional experiments and therefore need only small amounts of spectrometer time. The performance of the experiments is demonstrated with the application to two protein domains.

  4. MUSIC in triple-resonance experiments: amino acid type-selective (1)H-(15)N correlations

    Schubert; Smalla; Schmieder; Oschkinat

    1999-11-01

    Amino acid type-selective triple-resonance experiments can be of great help for the assignment of protein spectra, since they help to remove ambiguities in either manual or automated assignment procedures. Here, modified triple-resonance experiments that yield amino acid type-selective (1)H-(15)N correlations are presented. They are based on novel coherence transfer schemes, the MUSIC pulse sequence elements, that replace the initial INEPT transfer and are selective for XH(2) or XH(3) (X can be (15)N or (13)C). The desired amino acid type is thereby selected based on the topology of the side chain. Experiments for Gly (G-HSQC); Ala (A-HSQC); Thr, Val, Ile, and Ala (TAVI-HSQC); Thr and Ala (TA-HSQC), as well as Asn and Gln (N-HSQC and QN-HSQC), are described. The new experiments are recorded as two-dimensional experiments and therefore need only small amounts of spectrometer time. The performance of the experiments is demonstrated with the application to two protein domains. Copyright 1999 Academic Press. PMID:10527741

  5. 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.

  6. 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.

  7. (1)H, (13)C and (15)N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    Verma, Deepshikha; Bhattacharya, Alok; Chary, Kandala V R

    2016-04-01

    We report almost complete sequence specific (1)H, (13)C and (15)N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization. PMID:26377206

  8. (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

  9. 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.

  10. 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

  11. Determination of level widths in 15N using nuclear resonance fluorescence

    Szücs T.

    2015-01-01

    Full Text Available Level widths in 15N have been measured with the nuclear resonance fluorescence (NRF technique. Solid nitrogen compounds, bremsstrahlung, and HPGe detectors have been used as target, beam, and detectors, respectively. The preliminarily level widths are in agreement with the literature values, but more precise.

  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. RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

    Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E. [National Magnetic Resonance Facility at Madison (United States); Eghbalnia, Hamid R., E-mail: eghbalhd@uc.edu [University of Cincinnati, Department of Molecular and Cellular Physiology (United States)

    2012-04-15

    The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ({sup 1}H-{sup 15}N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ({sup 1}H-{sup 1}H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino

  14. 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.

  15. Study of the giant dipole resonances of 16O and 15N by means of radiative captures

    The giant dipole resonance in 16O and 15N is studied with reactions 14N(d,γ0)16O, 13C(3He,γ0)16O and 11B(α,γ0)15N. The same energy range is observed with transfert reactions as 12C(7Li,αγ)15N. A comparative study of radiative captures leading to 16O and 15N point out the importance of nsub(p)-nsub(t) configurations. Apparatus and experimental techniques developed are also described

  16. 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.

  17. 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....

  18. Amino acid selective unlabeling for sequence specific resonance assignments in proteins

    Krishnarjuna, B.; Jaipuria, Garima; Thakur, Anushikha [Indian Institute of Science, NMR Research Centre (India); D' Silva, Patrick, E-mail: patrick@biochem.iisc.ernet.in [Indian Institute of Science, Department of Biochemistry (India); Atreya, Hanudatta S., E-mail: hsatreya@sif.iisc.ernet.in [Indian Institute of Science, NMR Research Centre (India)

    2011-01-15

    Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective 'unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly {sup 13}C/{sup 15}N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {l_brace}{sup 12}CO{sub i}-{sup 15}N{sub i+1}{r_brace}-filtered HSQC, which aids in linking the {sup 1}H{sup N}/{sup 15}N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to {sup 2}H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of {sup 14}N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

  19. A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system

    For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized 15N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the 1H-15N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in 1H-15N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in 1H-15N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.Abbreviation: HSQC - heteronuclear single quantum coherence spectroscopy

  20. Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments

    Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as 13C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel 15N direct-detection experiments. The CAN experiment sequentially connects amide 15N resonances using 13Cα chemical shift matching, and the CON experiment connects the preceding 13C' nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding 13C-detecting experiment, NCA-DIPAP or NCO-IPAP (Bermel et al. 2006b; Takeuchi et al. 2008). However, the disadvantage due to the lower γ is counteracted by the slower 15N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the 15N observe CAN experiment is 16% higher than in the 13C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field Cα resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D2O. Thus, these features and the superior resolution of 15N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.

  1. NMR Assignment of Polymerase β labeled with 2H, 13C, and 15N in complex with substrate DNA

    Mueller, Geoffrey A.; DeRose, Eugene F.; Kirby, Thomas W.; London, Robert E.

    2007-01-01

    DNA Polymerase β is a multifunctional enzyme involved in base excision repair of nuclear DNA in vertebrate cells. It has been extensively studied as a model for mechanistic studies of the nucleotidyl transferase reaction, DNA synthesis fidelity, and protein-DNA interactions. Previous studies of 13C-methyl-methionine labeled Rat pol β revealed extensive dynamics in response to various DNA repair substrates (Bose-Basu et. al, 2004). We present here the first assignments of the full-length prote...

  2. Triple-Resonance Methods for Complete Resonance Assignment of Aromatic Protons and Directly Bound Heteronuclei in Histidine and Tryptophan Residues

    A set of three experiments is described which correlate aromatic resonances of histidine and tryptophan residues with amide resonances in 13C/15N-labelled proteins. Provided that backbone 1H and 15N positions of the sequentially following residues are known, this results in sequence-specific assignment of histidine chemical shifts. In the reverse situation, these residues can be located in the 1H-15N correlation map to facilitate backbone assignments. It may be chosen between selective versions for either of the two amino acid types or simultaneous detection of both with complete discrimination against phenylalanine or tyrosine residues in each case. The linkages between δ-proton/carbon and the remaining aromatic as well as backbone resonances do not rely on through-space interactions, which may be ambiguous, but exclusively employ one-bond scalar couplings for magnetization transfer instead. Knowledge of these aromatic chemical shifts is the prerequisite for the analysis of NOESY spectra, the study of protein-ligand interactions involving histidine and tryptophan residues and the monitoring of imidazole protonation states during pH titrations. The new methods are demonstrated with five different proteins with molecular weights ranging from 11 to 28 kDa

  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. Absolute hydrogen depth profiling using the resonant $^{1}$H($^{15}$N,$\\alpha\\gamma$)$^{12}$C nuclear reaction

    Reinhardt, Tobias P; Bemmerer, Daniel; Stöckel, Klaus; Wagner, Louis

    2016-01-01

    Resonant nuclear reactions are a powerful tool for the determination of the amount and profile of hydrogen in thin layers of material. Usually, this tool requires the use of a standard of well-known composition. The present work, by contrast, deals with standard-less hydrogen depth profiling. This approach requires precise nuclear data, e.g. on the widely used $^{1}$H($^{15}$N,$\\alpha\\gamma$)$^{12}$C reaction, resonant at 6.4\\,MeV $^{15}$N beam energy. Here, the strongly anisotropic angular distribution of the emitted $\\gamma$-rays from this resonance has been re-measured, resolving a previous discrepancy. Coefficients of (0.38$\\pm$0.04) and (0.80$\\pm$0.04) have been deduced for the second and fourth order Legendre polynomials, respectively. In addition, the resonance strength has been re-evaluated to (25.0$\\pm$1.5)\\,eV, 10\\% higher than previously reported. A simple working formula for the hydrogen concentration is given for cases with known $\\gamma$-ray detection efficiency. Finally, the absolute approach i...

  5. Absolute hydrogen depth profiling using the resonant 1H(15N, αγ)12C nuclear reaction

    Reinhardt, Tobias P.; Akhmadaliev, Shavkat; Bemmerer, Daniel; Stöckel, Klaus; Wagner, Louis

    2016-08-01

    Resonant nuclear reactions are a powerful tool for the determination of the amount and profile of hydrogen in thin layers of material. Usually, this tool requires the use of a standard of well-known composition. The present work, by contrast, deals with standard-less hydrogen depth profiling. This approach requires precise nuclear data, e.g. on the widely used 1 H(15 N, αγ)12 C reaction, resonant at 6.4 MeV 15 N beam energy. Here, the strongly anisotropic angular distribution of the emitted γ -rays from this resonance has been re-measured, resolving a previous discrepancy. Coefficients of (0.38 ± 0.04) and (0.80 ± 0.04) have been deduced for the second and fourth order Legendre polynomials, respectively. In addition, the resonance strength has been re-evaluated to (25.0 ± 1.5) eV, 10% higher than previously reported. A simple working formula for the hydrogen concentration is given for cases with known γ -ray detection efficiency. Finally, the absolute approach is illustrated using two examples.

  6. Ratios of 15N/12C and 4He/12C inclusive electroproduction cross sections in the nucleon resonance region

    Bosted, P E; Amarian, M; Anefalos, S; Anghinolfi, M; Asryan, G; Avakian, H; Bagdasaryan, H; Baillie, N; Ball, J P; Baltzell, N A; Barrow, S; Batourine, V; Battaglieri, M; Beard, K; Bedlinskiy, I; Bektasoglu, M; Bellis, M; Benmouna, N; Biselli, A S; Bonner, B E; Bouchigny, S; Boiarinov, S; Bradford, R; Branford, D; Brooks, W K; Bültmann, S; Burkert, V D; Butuceanu, C; Calarco, J R; Careccia, S L; Carman, D S; Carnahan, B; Cazes, A; Chen, S; Cole, P L; Collins, P; Coltharp, P; Cords, D; Corvisiero, P; Crabb, D; Crannell, H; Credé, V; Cummings, J P; De Masi, R; De Vita, R; De Sanctis, E; Degtyarenko, P V; Denizli, H; Dennis, L; Deur, A; Djalali, C; Dodge, G E; Donnelly, J; Doughty, D; Dragovitsch, P; Dugger, M; Dharmawardane, K V; Dytman, S; Dzyubak, O P; Egiyan, H; Egiyan, K S; Elouadrhiri, L; Eugenio, P; Fatemi, R; Fedotov, G; Feuerbach, R J; Forest, T A; Fradi, A; Funsten, H; Garçon, M; Gavalian, G; Gilfoyle, G P; Giovanetti, K L; Girod, F X; Goetz, J T; Golovatch, E; Gothe, R W; Griffioen, K A; Guidal, M; Guillo, M; Guler, N; Guo, L; Gyurjyan, V; Hadjidakis, C; Hafidi, K; Hakobyan, R S; Hardie, J; Heddle, D; Hersman, F W; Hicks, K; Hleiqawi, I; Holtrop, M; Huertas, M; Hyde-Wright, C E; Ilieva, Y; Ireland, D G; Ishkhanov, B S; Isupov, E L; Ito, M M; Jenkins, D; Jo, H S; Joo, K; Jüngst, H G; Kalantarians, N; Keith, C; Kellie, J D; Khandaker, M; Kim, K Y; Kim, K; Kim, W; Klein, A; Klein, F J; Klusman, M; Kossov, M; Kramer, L H; Kubarovski, V; Kühn, J; Kuhn, S E; Kuleshov, S V; Lachniet, J; Laget, J M; Langheinrich, J; Lawrence, D; Ji Li; Lima, A C S; Livingston, K; Lü, H; Lukashin, K; MacCormick, M; Markov, N; McAleer, S; McKinnon, B; McNabb, J W C; Mecking, B A; Mestayer, M D; Meyer, C A; Mibe, T; Mikhailov, K; Minehart, R; Mirazita, M; Miskimen, R; Mokeev, V; Morand, L; Morrow, S A; Moteabbed, M; Müller, J; Mutchler, G S; Nadel-Turonski, P; Nasseripour, R; Niccolai, S; Niculescu, G; Niculescu, I; Niczyporuk, B B; Niroula, M R; Niyazov, R A; Nozar, M; O'Rielly, G V; Osipenko, M; Ostrovidov, A I; Park, K; Pasyuk, E; Paterson, C; Philips, S A; Pierce, J; Pivnyuk, N; Pocanic, D; Pogorelko, O; Polli, E; Pozdniakov, S; Preedom, B M; Price, J W; Prok, Y; Protopopescu, D; Qin, L M; Raue, B A; Riccardi, G; Ricco, G; Ripani, M; Rosner, G; Rossi, P; Rowntree, D; Rubin, P D; Sabati, F; Salgado, C; Santoro, J P; Sapunenko, V; Schumacher, R A; Serov, V S; Sharabyan, Yu G; Shaw, J; Shvedunov, N V; Skabelin, A V; Smith, E S; Smith, L C; Sober, D I; Stavinsky, A; Stepanyan, S S; Stepanyan, S; Stokes, B E; Stoler, P; Strauch, S; Suleiman, R; Taiuti, M; Taylor, S; Tedeschi, D J; Thoma, U; Tkabladze, A; Tkachenko, S; Todor, L; Ungaro, M; Vineyard, M F; Vlassov, A V; Weinstein, L B; Weygand, D P; Williams, M; Wolin, E; Wood, M H; Yegneswaran, A; Yun, J; Zana, L; Zhang, J; Zhao, B; Zhao, Z

    2007-01-01

    The ratio of inclusive electron scattering cross sections for 15N/12C was determined in the kinematic range 0.8resonance structure, as predicted by a phenomenological model, and also by quark-hadron duality. Within the super-scaling quasi-elastic model, slight evidence is found for a 1 MeV lower effective nucleon binding energy in 15N than in 12C. Ratios of 4He/12C using 1.6 to 2.5 GeV electrons are in good agreement with the phenomenological model.

  7. 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

  8. An expectation/maximization nuclear vector replacement algorithm for automated NMR resonance assignments

    Langmead, Christopher James [Dartmouth Computer Science Department (United States); Donald, Bruce Randall [Dartmouth Center for Structural Biology and Computational Chemistry (United States)], E-mail: brd@cs.dartmouth.edu

    2004-06-15

    We report an automated procedure for high-throughput NMR resonance assignment for a protein of known structure, or of an homologous structure. Our algorithm performs Nuclear Vector Replacement (NVR) by Expectation/Maximization (EM) to compute assignments. NVR correlates experimentally-measured NH residual dipolar couplings (RDCs) and chemical shifts to a given a priori whole-protein 3D structural model. The algorithm requires only uniform {sup 15}N-labelling of the protein, and processes unassigned H{sup N}-{sup 15}N HSQC spectra, H{sup N}-{sup 15}N RDCs, and sparse H{sup N}-H{sup N} NOE's (d{sub NN}s). NVR runs in minutes and efficiently assigns the (H{sup N},{sup 15}N) backbone resonances as well as the sparse d{sub NN}s from the 3D {sup 15}N-NOESY spectrum, in O(n{sup 3}) time. The algorithm is demonstrated on NMR data from a 76-residue protein, human ubiquitin, matched to four structures, including one mutant (homolog), determined either by X-ray crystallography or by different NMR experiments (without RDCs). NVR achieves an average assignment accuracy of over 99%. We further demonstrate the feasibility of our algorithm for different and larger proteins, using different combinations of real and simulated NMR data for hen lysozyme (129 residues) and streptococcal protein G (56 residues), matched to a variety of 3D structural models. Abbreviations: NMR, nuclear magnetic resonance; NVR, nuclear vector replacement; RDC, residual dipolar coupling; 3D, three-dimensional; HSQC, heteronuclear single-quantum coherence; H{sup N}, amide proton; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; d{sub NN}, nuclear Overhauser effect between two amide protons; MR, molecular replacement; SAR, structure activity relation; DOF, degrees of freedom; nt., nucleotides; SPG, Streptococcal protein G; SO(3), special orthogonal (rotation) group in 3D; EM, Expectation/Maximization; SVD, singular value decomposition.

  9. Towards Automated Structure-Based NMR Resonance Assignment

    Jang, Richard; Gao, Xin; Li, Ming

    We propose a general framework for solving the structure-based NMR backbone resonance assignment problem. The core is a novel 0-1 integer programming model that can start from a complete or partial assignment, generate multiple assignments, and model not only the assignment of spins to residues, but also pairwise dependencies consisting of pairs of spins to pairs of residues. It is still a challenge for automated resonance assignment systems to perform the assignment directly from spectra without any manual intervention. To test the feasibility of this for structure-based assignment, we integrated our system with our automated peak picking and sequence-based resonance assignment system to obtain an assignment for the protein TM1112 with 91% recall and 99% precision without manual intervention. Since using a known structure has the potential to allow one to use only N-labeled NMR data and avoid the added expense of using C-labeled data, we work towards the goal of automated structure-based assignment using only such labeled data. Our system reduced the assignment error of Xiong-Pandurangan-Bailey-Kellogg's contact replacement (CR) method, which to our knowledge is the most error-tolerant method for this problem, by 5 folds on average. By using an iterative algorithm, our system has the added capability of using the NOESY data to correct assignment errors due to errors in predicting the amino acid and secondary structure type of each spin system. On a publicly available data set for Ubiquitin, where the type prediction accuracy is 83%, we achieved 91% assignment accuracy, compared to the 59% accuracy that was obtained without correcting for typing errors.

  10. Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States

    A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cγ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues

  11. 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...

  12. Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data

    Zeng Jianyang [Duke University, Department of Computer Science (United States); Zhou Pei [Duke University Medical Center, Department of Biochemistry (United States); Donald, Bruce Randall [Duke University, Department of Computer Science (United States)

    2011-08-15

    One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called Nasca (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), Nasca extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that Nasca assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by Nasca have backbone RMSD 0

  13. Exploiting image registration for automated resonance assignment in NMR

    Analysis of protein NMR data involves the assignment of resonance peaks in a number of multidimensional data sets. To establish resonance assignment a three-dimensional search is used to match a pair of common variables, such as chemical shifts of the same spin system, in different NMR spectra. We show that by displaying the variables to be compared in two-dimensional plots the process can be simplified. Moreover, by utilizing a fast Fourier transform cross-correlation algorithm, more common to the field of image registration or pattern matching, we can automate this process. Here, we use sequential NMR backbone assignment as an example to show that the combination of correlation plots and segmented pattern matching establishes fast backbone assignment in fifteen proteins of varying sizes. For example, the 265-residue RalBP1 protein was 95.4 % correctly assigned in 10 s. The same concept can be applied to any multidimensional NMR data set where analysis comprises the comparison of two variables. This modular and robust approach offers high efficiency with excellent computational scalability and could be easily incorporated into existing assignment software

  14. 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

  15. 15N solid-state nuclear magnetic resonance study of pyrolyzed metal-polyaniline cathode catalysts for oxygen reduction in fuel cells

    Kuroki, Shigeki; Hosaka, Yo; Yamauchi, Chiharu; Nagata, Shinsuke; Sonoda, Mayu

    2015-09-01

    The oxygen reduction reaction (ORR) activity of pyrolyzed metal-free and metal (Mn, Fe, Co, Ni and Cu)-containing polyaniline (PANI) in polymer electrolyte fuel cell (PEFC) was studied. The metal-free PANI800 shows quite poor ORR catalytic activity, whilst the metal-containing PANIMe800 display a better ORR activity. The 15N CP/MAS NMR spectra of PANINi800 and PANICu800 show one weak peak at 118 ppm and there is no peak observed in PANIFe800, against that of PANI800, PANIMn800, PANICo800 and PANINi800 show two peaks at 273 and 118 ppm assigned to the pyridinic and pyridinium nitrogens. It is because of the paramagnetic effect of metal ions. The 15N spin-echo NMR spectra of PANIMe800 with fast recycle delay show the peaks at 140 and 270 ppm assigned to the graphitic and pyridinic nitrogens, against that of PANI800 shows no peak. The spectra of PANIMn800, PANICo800, PANINi800 and PANICu600 also contain a very broaden peak at 430 ppm assigned to the nitrogen with Fermi-contact effect from metal ions. The spectra of PANIFe800 show some spinning side bands and the average Fe3+-15N distance can be calculated. The some amount of iron ion are relieved and average Fe3+-15N distance increase after acid washing and the ORR activity decreases.

  16. Assignment of the side-chain 1H and 13C resonances of interleukin-1β using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

    The assignment of the aliphatic 1H and 13C resonances of IL-1β, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13Cα chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13Cα (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13Cα(i) and some weaker interresidue NH(i)-15N(i)-Cα(i-1) correlations, the former via intraresidue one-bond 1JNCα and the latter via interresidue two-bond 2HNCα couplings. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. The authors were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained

  17. 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.

  18. A novel way of amino acid-specific assignment in {sup 1}H-{sup 15}N HSQC spectra with a wheat germ cell-free protein synthesis system

    Morita, Eugene Hayato, E-mail: ehmorita@dpc.ehime-u.ac.jp; Shimizu, Masato [Ehime University, Division of Gene Research, Department of Molecular Science, Integrated Center for Science (Japan); Ogasawara, Tomio; Endo, Yaeta; Tanaka, Rikou; Kohno, Toshiyuki [Mitsubishi Kagaku Institute of Life Sciences (MITILS) (Japan)], E-mail: tkohno@ibra.ls.m-kagaku.co.jp

    2004-09-15

    For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized {sup 15}N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the {sup 1}H-{sup 15}N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in {sup 1}H-{sup 15}N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in {sup 1}H-{sup 15}N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.Abbreviation: HSQC - heteronuclear single quantum coherence spectroscopy.

  19. Resonance strengths in the 14N(p,gamma)15O and 15N(p,alpha gamma)12C reactions

    Marta, Michele; Bemmerer, Daniel; Beyer, Roland; Broggini, Carlo; Caciolli, Antonio; Erhard, Martin; Fülöp, Zsolt; Grosse, Eckart; Gyürky, György; Hannaske, Roland; Junghans, Arnd R; Menegazzo, Roberto; Nair, Chithra; Schwengner, Ronald; Szücs, Tamás; Vezzú, Simone; Wagner, Andreas; Yakorev, Dmitry

    2010-01-01

    The 14N(p,gamma)15O reaction is the slowest reaction of the carbon-nitrogen-oxygen cycle of hydrogen burning in stars. As a consequence, it determines the rate of the cycle. The 15N(p,alpha gamma)12C reaction is frequently used in inverse kinematics for hydrogen depth profiling in materials. The 14N(p,gamma)15O and 15N(p,alpha gamma)12C reactions have been studied simultaneously, using titanium nitride targets of natural isotopic composition and a proton beam. The strengths of the resonances at Ep = 1058 keV in 14N(p,gamma)15O and at Ep = 897 and 430 keV in 15N(p,alpha gamma)12C have been determined with improved precision, relative to the well-known resonance at Ep = 278 keV in 14N(p,gamma)15O. The new recommended values are \\omega\\gamma = 0.352$\\pm$0.018, 362$\\pm$20, and 22.0$\\pm$0.9\\,eV for their respective strengths. In addition, the branching ratios for the decay of the Ep = 1058 keV resonance in 14N(p,gamma)15O have been redetermined. The data reported here should facilitate future studies of off-resona...

  20. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis.

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian; Ulrich, Anne S

    2015-06-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  1. Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy

    We report here the backbone 1HN, 15N, 13Cα, 13CO, and 1Hα NMR assignments for the catalytic domain of human fibroblast collagenase (HFC). Three independent assignment pathways (matching 1H, 13Cα, and 13CO resonances) were used to establish sequential connections. The connections using 13Cα resonances were obtained from HNCOCA and HNCA experiments; 13CO connections were obtained from HNCO and HNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and 15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement with recent X-ray structures of human fibroblast collagenase and human neutrophil collagenase as well as similar to recent NMR structures of a highly homologous protein, stromelysin. All three helices were located; a five-stranded β-sheet (four parallel strands, one antiparallel strand) was also determined. β-Sheet regions were identified by cross-strand dαN and dNN connections and by strong intraresidue dαN correlations, and were corroborated by observing slow amide proton exchange. Chemical shift changes in a selectively 15N-labeled sample suggest that substantial structural changes occur in the active site cleft on the binding of an inhibitor

  2. (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

  3. Complete assignment of the methionyl carbonyl carbon resonance in switch variant anti-dansyl antibodies labeled with (1- sup 13 C)methionine

    Kato, Koichi; Matsunaga, C.; Igarashi, Takako; Kim, Hahyung; Odaka, Asano; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo, Hongo (Japan))

    1991-01-01

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire C{sub H}1 domain is deleted. The switch variant antibodies were specifically labeled with (1-{sup 13}C)methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating {sup 15}N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of ({alpha}-{sup 15}N)lysine and ({sup 15}N)threonine, both of which cannot become the substrate of transaminases. It was found that {beta}-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with {sup 15}N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val. On the basis of the results of the present {sup 13}C study, possible use of the assigned carbonyl carbon resonances for the elucidation of the structure-function relationship in the antibody system has been briefly discussed.

  4. Vibrational Assignments of Six-Coordinate Ferrous Heme Nitrosyls: New Insight From Nuclear Resonance Vibrational Spectroscopy

    Paulat, F.; Berto, T.C.; George, S.DeBeer; Goodrich, L.; Praneeth, V.K.K.; Sulok, C.D.; Lehnert, N.

    2009-05-21

    This Communication addresses a long-standing problem: the exact vibrational assignments of the low-energy modes of the Fe-N-O subunit in six-coordinate ferrous heme nitrosyl model complexes. This problem is addressed using nuclear resonance vibrational spectroscopy (NRVS) coupled to {sup 15}N{sup 18}O isotope labeling and detailed simulations of the obtained data. Two isotope-sensitive features are identified at 437 and 563 cm{sup -1}. Normal coordinate analysis shows that the 437 cm{sup -1} mode corresponds to the Fe-NO stretch, whereas the 563 cm{sup -1} band is identified with the Fe-N-O bend. The relative NRVS intensities of these features determine the degree of vibrational mixing between the stretch and the bend. The implications of these results are discussed with respect to the trans effect of imidazole on the bound NO. In addition, a comparison to myoglobin-NO (Mb-NO) is made to determine the effect of the Mb active site pocket on the bound NO.

  5. Efficient assignment of methyl resonances: Enhanced sensitivity by gradient selection in a DE-MQ-(H)CCmHt m-TOCSY experiment

    We present a gradient selected and doubly sensitivity-enhanced DE-MQ-(H)CCmHm-TOCSY experiment for the sequence-specific assignment of methyl resonances in 13C,15N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the 13Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under 13C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using 15N,13C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 deg. D2O. A 16-h experiment on 600 MHz 1H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies

  6. MONTE: An automated Monte Carlo based approach to nuclear magnetic resonance assignment of proteins

    Hitchens, T. Kevin; Lukin, Jonathan A.; Zhan Yiping; McCallum, Scott A.; Rule, Gordon S. [Carnegie Mellon University, Department of Biological Sciences (United States)], E-mail: rule@andrew.cmu.edu

    2003-01-15

    A general-purpose Monte Carlo assignment program has been developed to aid in the assignment of NMR resonances from proteins. By virtue of its flexible data requirements the program is capable of obtaining assignments of both heavily deuterated and fully protonated proteins. A wide variety of source data, such as inter-residue scalar connectivity, inter-residue dipolar (NOE) connectivity, and residue specific information, can be utilized in the assignment process. The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. The assignment of a deuterated 45 kDa homodimeric Glutathione-S-transferase illustrates the principal features of the program.

  7. 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.

  8. 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)

  9. Applications of the 18O-isotope shift on 13C and 15N nuclear magnetic resonance spectroscopy to the study of bioorganic reaction mechanisms

    The study of reactions involving the formation and cleavage of carbon-oxygen or nitrogen-oxygen bonds has been significantly aided by recent demonstrations of the generality and characteristics of the 18O-isotope shift in 13C and 15N nuclear magnetic resonance spectroscopy. In many instances, the magnitudes of the 18O-induced isotopic shifts are sufficiently large as to permit the use of even modest NMR instrumentation and natural abundance 13C. Studies involving less soluble compounds, higher molecular weight materials or relatively rapid reactions may often be carried out using 13C enrichment. Because NMR spectroscopy is non-destructive, it has proven to be extremely useful in the study of natural product biosynthetic pathways. Another area where important applications are being made is in the study of enzymatic and non-enzymatic reaction mechanisms. The characteristics of the 18O isotope shift in 13C NMR spectroscopy are reviewed. Several examples from the work of other groups in the area of natural product biosynthesis are briefly mentioned. This is followed by a number of illustrative applications in the area of bioorganic and enzymatic reaction mechanism that have been examined in our laboratory. The enzymatic examples include acid phosphatases, epoxide hydratase, acetylcholinesterase and asparaginase. 20 refs.; 1 figure

  10. λ cro repressor complex with O/sub R/3 DNA: 15N NMR observations

    15N NMR studies of the coliphage λ cro repressor are presented. The protein has been uniformly labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein it has been proposed that the C-terminus is involved in DNA binding. These experiments give direct verification of that proposal. [15N] Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N{1H} nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/J/sub NH/(10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested and which would significantly affect the properties of the C-terminal arm, is shown to not occur

  11. 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.

  12. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme

    Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the charge-relay system or catalytic triad of serine proteases. There have been a number of reports that have cast doubt on its correctness. In the present work the authors have tested this assignment using α-lytic protease, a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N/sup delta1/ of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N-/sup delta1/-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal

  13. An automated framework for NMR resonance assignment through simultaneous slice picking and spin system forming

    Abbas, Ahmed

    2014-04-19

    Despite significant advances in automated nuclear magnetic resonance-based protein structure determination, the high numbers of false positives and false negatives among the peaks selected by fully automated methods remain a problem. These false positives and negatives impair the performance of resonance assignment methods. One of the main reasons for this problem is that the computational research community often considers peak picking and resonance assignment to be two separate problems, whereas spectroscopists use expert knowledge to pick peaks and assign their resonances at the same time. We propose a novel framework that simultaneously conducts slice picking and spin system forming, an essential step in resonance assignment. Our framework then employs a genetic algorithm, directed by both connectivity information and amino acid typing information from the spin systems, to assign the spin systems to residues. The inputs to our framework can be as few as two commonly used spectra, i.e., CBCA(CO)NH and HNCACB. Different from the existing peak picking and resonance assignment methods that treat peaks as the units, our method is based on \\'slices\\', which are one-dimensional vectors in three-dimensional spectra that correspond to certain (N, H) values. Experimental results on both benchmark simulated data sets and four real protein data sets demonstrate that our method significantly outperforms the state-of-the-art methods while using a less number of spectra than those methods. Our method is freely available at http://sfb.kaust.edu.sa/Pages/Software.aspx. © 2014 Springer Science+Business Media.

  14. 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 ...

  15. Automated solid-state NMR resonance assignment of protein microcrystals and amyloids

    Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218–289) and α-synuclein yielded 88–97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77–90 % correctness if also assignments classified as tentative by the algorithm are included

  16. Automated solid-state NMR resonance assignment of protein microcrystals and amyloids

    Schmidt, Elena [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany); Gath, Julia [ETH Zurich, Physical Chemistry (Switzerland); Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Ravotti, Francesco; Szekely, Kathrin; Huber, Matthias [ETH Zurich, Physical Chemistry (Switzerland); Buchner, Lena [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Guentert, Peter, E-mail: guentert@em.uni-frankfurt.de [Goethe University Frankfurt am Main, Center for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry (Germany)

    2013-07-15

    Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218-289) and {alpha}-synuclein yielded 88-97 % correctness for the backbone and side-chain assignments that are classified as self-consistent by the algorithm, and 77-90 % correctness if also assignments classified as tentative by the algorithm are included.

  17. EZ-ASSIGN, a program for exhaustive NMR chemical shift assignments of large proteins from complete or incomplete triple-resonance data

    For several of the proteins in the BioMagResBank larger than 200 residues, 60 % or fewer of the backbone resonances were assigned. But how reliable are those assignments? In contrast to complete assignments, where it is possible to check whether every triple-resonance Generalized Spin System (GSS) is assigned once and only once, with incomplete data one should compare all possible assignments and pick the best one. But that is not feasible: For example, for 200 residues and an incomplete set of 100 GSS, there are 1.6 × 10260 possible assignments. In “EZ-ASSIGN”, the protein sequence is divided in smaller unique fragments. Combined with intelligent search approaches, an exhaustive comparison of all possible assignments is now feasible using a laptop computer. The program was tested with experimental data of a 388-residue domain of the Hsp70 chaperone protein DnaK and for a 351-residue domain of a type III secretion ATPase. EZ-ASSIGN reproduced the hand assignments. It did slightly better than the computer program PINE (Bahrami et al. in PLoS Comput Biol 5(3):e1000307, 2009) and significantly outperformed SAGA (Crippen et al. in J Biomol NMR 46:281–298, 2010), AUTOASSIGN (Zimmerman et al. in J Mol Biol 269:592–610, 1997), and IBIS (Hyberts and Wagner in J Biomol NMR 26:335–344, 2003). Next, EZ-ASSIGN was used to investigate how well NMR data of decreasing completeness can be assigned. We found that the program could confidently assign fragments in very incomplete data. Here, EZ-ASSIGN dramatically outperformed all the other assignment programs tested

  18. A program for semi-automatic sequential resonance assignments in protein 1H nuclear magnetic resonance spectra

    Billeter, M.; Basus, V. J.; Kuntz, I. D.

    A new approach to the sequential resonance assignment of protein 1H NMR spectra based on a computer program is presented. Two main underlying concepts were used in the design of this program. First, it considers at any time all possible assignments that are consistent with the currently available data. If new information is added then assignments that have become inconsistent are eliminated. Second, the process of the assignment is split into formal steps that follow strictly from the available data and steps that involve the interpretation of ambiguous NMR data. The first kind of step is safe in the sense that it never leads to false assignments provided that the input does not contain any error; these steps are executed automatically by the program when the input files are read and whenever new data have been entered interactively. The second kind of step is left to the user: An interactive dialog provides detailed information on the current situation of the assignment and indicates what kind of new data would be most promising for further assignment. The user then provides new data to the program and restarts the automatic part which will attempt to draw logical conclusions from the joint use of the new data and the earlier available information and will eliminate assignments that have become inconsistent. Results of test problems using simulated NMR data for proteins consisting of up to 99 residues as well as the application of the program to obtain the complete assignment of α-bungarotoxin, a 74-residue snake neurotoxin, are reported.

  19. {sup 1}H and {sup 15}N NMR assignment and solution structure of the SH3 domain of spectrin: Comparison of unrefined and refined structure sets with the crystal structure

    Blanco, Francisco J.; Ortiz, Angel R.; Serrano, Luis [European Molecular Biology Laboratory (Germany)

    1997-06-15

    The assignment of the {sup 1}H and {sup 15}Nnuclear magnetic resonance spectra of the Src-homology region 3 domain of chicken brain {alpha}-spectrin has been obtained. A set of solution structures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure in solution, as evaluated by a principal component analysis of the global pairwise root-mean-square deviation (rmsd) in a large set of structures consisting of the refined and unrefined solution structures and the crystal structure. The solution structure is well defined, with a lower degree of convergence between the structures in the loop regions than in the secondary structure elements. The average pairwise rmsd between the 15 refined solution structures is 0.71 {+-} 0.13 A for the backbone atoms and 1.43 {+-} 0.14 A for all heavy atoms. The solution structure is basically the same as the crystal structure. The average rmsd between the 15 refined solution structures and the crystal structure is 0.76 A for the backbone atoms and 1.45 {+-} 0.09 A for all heavy atoms. There are, however, small differences probably caused by intermolecular contacts in the crystal structure.

  20. 4D experiments measured with APSY for automated backbone resonance assignments of large proteins

    Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40 kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a ‘just-in-time’ manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90 % of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4 kDa.

  1. Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: Sequential assignment of resonances

    The authors report the sequential assignment of resonances to specific residues in the proton nuclear magnetic resonance spectrum of the variant-3 neurotoxin from the scorpion Centruroides sculpturatus Ewing (range southwestern U.S.A.). A combination of two-dimensional NMR experiments such as 2D-COSY, 2D-NOESY, and single- and double-RELAY coherence transfer spectroscopy has been employed on samples of the protein dissolved in D2O and in H2O for assignment purposes. These studies provide a basis for the determination of the solution-phase conformation of this protein and for undertaking detailed structure-function studies of these neurotoxins that modulate the flow of sodium current by binding to the sodium channels of excitable membranes

  2. A novel strategy for NMR resonance assignment and protein structure determination

    The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution – especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.

  3. Sequence determination and resonance assignments of an Azomonas siderophore using 13C natural abundance 13C-1H HNCA experiment

    Wasielewski, E.; Abdallah, M. A.; Kyslík, Pavel; Kieffer, B.

    2001-01-01

    Roč. 4, - (2001), s. 765-770. ISSN 1387-1609 Institutional research plan: CEZ:AV0Z5020903 Keywords : determination * resonance * assignments Subject RIV: EE - Microbiology, Virology Impact factor: 0.555, year: 2001

  4. Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

    The chemical shift assignments and secondary structure of a murine-human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on a uniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basis of chemical shifts, NH-CαH coupling constants, medium-range NOEs and the location of slowly exchanging amide protons. The protein contains four α-helices, the relative orientations of which were determined on the basis of long-range, interhelical NOEs. The four helices are arranged in an up-up-down-down orientation, as found in other four-helical bundle cytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographic structure for murine LIF [Robinson et al. (1994) Cell, 77, 1101-1116]. Differences between the X-ray structure and the solution structure are evident in the N-terminal tail, where the solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystal structure and the small antiparallel β-sheet encompassing residues in the N-terminus and CD loop in the crystal structure is less stable

  5. Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon 13C NMR resonances in detergent-solubilized M13 coat protein

    The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. 13C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by 13C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both 13C and 15N. The carbonyl region of the natural-abundance 13C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pK/sub a/ of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K. Comparable spectra and digestion patterns were obtained when the protein was solubilized in sodium deoxycholate, suggesting that the coat protein binds both amphiphiles in a similar fashion

  6. Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry

    Scharff, A.M.; Egsgaard, H.; Hansen, P.E.;

    2003-01-01

    Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo N-15 nuclear magnetic resonance (NMR) by exposing detached nodules to N-15, via a perfusion medium, while recording a time course of spectra. In vivo P-31 NMR spectroscopy was used to monitor...... of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized N-15 gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible....

  7. Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively {sup 13}C-labelled proteins

    Higman, Victoria A. [Leibniz-Institut fuer Molekulare Pharmakologie (Germany); Flinders, Jeremy [Genentech, Inc., Structural Biology Department (United States); Hiller, Matthias; Jehle, Stefan; Markovic, Stefan; Fiedler, Sebastian; Rossum, Barth-Jan van; Oschkinat, Hartmut [Leibniz-Institut fuer Molekulare Pharmakologie (Germany)], E-mail: oschkinat@fmp-berlin.de

    2009-08-15

    In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-{sup 13}C]- and [2-{sup 13}C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [{sup 13}C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in {sup 13}C-{sup 13}C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken {alpha}-spectrin SH3 domain (62 residues), {alpha}B-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the C{alpha}, C{beta}, C' and N resonances in the core domain of {alpha}B-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein)

  8. Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively 13C-labelled proteins

    In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C' and N resonances in the core domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein)

  9. Assignment of amide proton signals by combined evaluation of HN, NN and HNCA MAS-NMR correlation spectra

    Rossum, Barth-Jan van; Castellani, Federica [Forschungsinstitut fuer Molekulare Pharmakologie (FMP) (Germany); Pauli, Jutta [BAM (Germany); Rehbein, Kristina [Forschungsinstitut fuer Molekulare Pharmakologie (FMP) (Germany); Hollander, J.; Groot, Huub J.M. de [BAM (Germany); Oschkinat, Hartmut [Forschungsinstitut fuer Molekulare Pharmakologie (FMP) (Germany)], E-mail: Oschkinat@fmp-berlin.de

    2003-03-15

    In this paper, we present a strategy for the {sup 1}H{sup N} resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the {alpha}-spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H{sup N}-N-C{sup {alpha}} correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D ({sup 1}H-{sup 15}N) and 3D ({sup 1}H-{sup 15}N-{sup 13}C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone {sup 15}N resonances and as an aid in the amide proton assignment.

  10. Selective excitation enables assignment of proton resonances and {sup 1}H-{sup 1}H distance measurement in ultrafast magic angle spinning solid state NMR spectroscopy

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy, E-mail: ramamoor@umich.edu [Biophysics and Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055 (United States)

    2015-07-21

    Remarkable developments in ultrafast magic angle spinning (MAS) solid-state NMR spectroscopy enabled proton-based high-resolution multidimensional experiments on solids. To fully utilize the benefits rendered by proton-based ultrafast MAS experiments, assignment of {sup 1}H resonances becomes absolutely necessary. Herein, we propose an approach to identify different proton peaks by using dipolar-coupled heteronuclei such as {sup 13}C or {sup 15}N. In this method, after the initial preparation of proton magnetization and cross-polarization to {sup 13}C nuclei, transverse magnetization of desired {sup 13}C nuclei is selectively prepared by using DANTE (Delays Alternating with Nutations for Tailored Excitation) sequence and then, it is transferred to bonded protons with a short-contact-time cross polarization. Our experimental results demonstrate that protons bonded to specific {sup 13}C atoms can be identified and overlapping proton peaks can also be assigned. In contrast to the regular 2D HETCOR experiment, only a few 1D experiments are required for the complete assignment of peaks in the proton spectrum. Furthermore, the finite-pulse radio frequency driven recoupling sequence could be incorporated right after the selection of specific proton signals to monitor the intensity buildup for other proton signals. This enables the extraction of {sup 1}H-{sup 1}H distances between different pairs of protons. Therefore, we believe that the proposed method will greatly aid in fast assignment of peaks in proton spectra and will be useful in the development of proton-based multi-dimensional solid-state NMR experiments to study atomic-level resolution structure and dynamics of solids.

  11. Resonance strengths in the 17,18O(p, α)14,15N reactions and background suppression underground. Commissioning of a new setup for charged-particle detection at LUNA

    We report on measurements of resonance strengths and energies for the Ep = 151 and 193 keV resonances in the 18O(p, α)15N and 17O(p, α)14N reactions, respectively, obtained during commissioning of a new setup for alpha-particle detection studies at the LUNA underground laboratory. Our values, ωγ(151) = 164.2 ± 0.9stat-11.7+12.1syst meV and ωγ (193) = 1.68 ± 0.03stat ± 0.12syst meV, are in excellent agreement with those reported in the literature. New values of resonance energies are Ep = 151.2 ± 0.3 keV and Ep = 194.8 ± 0.3 keV, respectively, this latter with the highest precision to date. Comparative background measurements in silicon detectors overground and underground were also carried out, yielding up to a factor of 15 in background suppression at LUNA at energies around 200 keV. This clearly demonstrates the usefulness of underground measurements in charged-particles experiments, especially at low detection energies. (orig.)

  12. Resonance strengths in the {sup 17,18}O(p, α){sup 14,15}N reactions and background suppression underground. Commissioning of a new setup for charged-particle detection at LUNA

    Bruno, C.G.; Scott, D.A.; Aliotta, M.; Davinson, T.; Griffin, C.J. [University of Edinburgh, SUPA, School of Physics and Astronomy, Edinburgh (United Kingdom); Formicola, A.; Best, A.; Junker, M. [Laboratori Nazionali del Gran Sasso, INFN, Assergi (Italy); Anders, M.; Bemmerer, D.; Szuecs, T. [Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden (Germany); Broggini, C.; Menegazzo, R. [INFN, Padova (Italy); Caciolli, A.; Depalo, R. [INFN, Padova (Italy); Universita di Padova, Dipartimento di Fisica e Astronomia, Padova (Italy); Cavanna, F.; Corvisiero, P.; Prati, P. [INFN, Genova (Italy); Dipartimento di Fisica, Universita di Genova, Genova (Italy); Di Leva, A.; Imbriani, G. [Universita di Napoli ' ' Federico II' ' , Dipartimento di Scienze Fisiche, Napoli (Italy); Elekes, Z.; Fueloep, Zs.; Gyuerky, Gy.; Somorjai, E. [MTA Atomki, Institute of Nuclear Research, Debrecen (Hungary); Gervino, G. [Universita degli Studi di Torino, Dipartimento di Fisica Sperimentale, Torino (Italy); Guglielmetti, A.; Trezzi, D. [Universita degli Studi di Milano (Italy); INFN, Milano (Italy); Gustavino, C. [INFN, Roma (Italy); Napolitani, E. [Universita di Padova, Dipartimento di Fisica e Astronomia, Padova (Italy); Straniero, O. [Laboratori Nazionali del Gran Sasso, INFN, Assergi (Italy); Osservatorio Astronomico di Collurania, Teramo (Italy); Strieder, F. [South Dakota School of Mines and Technology, Rapid City, SD (United States); Collaboration: LUNA Collaboration

    2015-08-15

    We report on measurements of resonance strengths and energies for the E{sub p} = 151 and 193 keV resonances in the {sup 18}O(p, α){sup 15}N and {sup 17}O(p, α){sup 14}N reactions, respectively, obtained during commissioning of a new setup for alpha-particle detection studies at the LUNA underground laboratory. Our values, ωγ(151) = 164.2 ± 0.9{sub stat-11.7}{sup +12.1}{sub syst} meV and ωγ (193) = 1.68 ± 0.03{sub stat} ± 0.12{sub syst} meV, are in excellent agreement with those reported in the literature. New values of resonance energies are E{sub p} = 151.2 ± 0.3 keV and E{sub p} = 194.8 ± 0.3 keV, respectively, this latter with the highest precision to date. Comparative background measurements in silicon detectors overground and underground were also carried out, yielding up to a factor of 15 in background suppression at LUNA at energies around 200 keV. This clearly demonstrates the usefulness of underground measurements in charged-particles experiments, especially at low detection energies. (orig.)

  13. Simultaneous acquisition of three NMR spectra in a single experiment for rapid resonance assignments in metabolomics

    Shivanand M Pudakalakatti; Abhinav Dubey; Hanudatta S Atreya

    2015-06-01

    NMR-based approach to metabolomics typically involves the collection of two-dimensional (2D) heteronuclear correlation spectra for identification and assignment of metabolites. In case of spectral overlap, a 3D spectrum becomes necessary, which is hampered by slow data acquisition for achieving sufficient resolution. We describe here a method to simultaneously acquire three spectra (one 3D and two 2D) in a single data set, which is based on a combination of different fast data acquisition techniques such as G-matrix Fourier transform (GFT) NMR spectroscopy, parallel data acquisition and non-uniform sampling. The following spectra are acquired simultaneously: (1) 13C multiplicity edited GFT (3,2)D HSQC-TOCSY, (2) 2D [1H-1H] TOCSY and (3) 2D [13C-1H] HETCOR. The spectra are obtained at high resolution and provide high-dimensional spectral information for resolving ambiguities. While the GFT spectrum has been shown previously to provide good resolution, the editing of spin systems based on their CH multiplicities further resolves the ambiguities for resonance assignments. The experiment is demonstrated on a mixture of 21 metabolites commonly observed in metabolomics. The spectra were acquired at natural abundance of 13C. This is the first application of a combination of three fast NMR methods for small molecules and opens up new avenues for high-throughput approaches for NMR-based metabolomics.

  14. A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

    Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHNα coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded β-sheet surrounded by four α-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine

  15. Spin and Parity Assignment of Neutron Resonances using Gamma-ray Multiplicity

    Decay gamma rays following neutron capture on various isotopes are collected by the Detector for Advanced Neutron Capture Experiments (DANCE) array, which is located at flight path 14 at the Lujan Neutron Scattering Center at Los Alamos National Laboratory. The high segmentation (160 detectors) and close packing of the detector array enable gamma-ray multiplicity measurements. The calorimetric properties of the DANCE array coupled with the neutron time-of-flight technique enables one to gate on a specific resonance of a given isotope in the time-of-flight spectrum and obtain the summed energy spectrum for that isotope. The singles gamma-ray spectrum for each multiplicity can be separated by their DANCE cluster multiplicity. The multiplicity distribution contains the signatures of spin and parity of the capture state. Under suitable circumstances where the difference between spins of the initial (capture) and final (ground) state is large enough, the signatures in the multiplicity distribution can be used in improving the spin assignment of the initial state. The spin assignment is applied with varying degree of success to difference isotopes and description of this application for 95Mo, 151,153Eu, and 155,157Gd is reviewed briefly.

  16. Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

    Several techniques for spectral editing of 2D 13C–13C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N–CO peaks through 13C–15N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH2) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other π-pulse is shifted from the center of a rotor period tr by about 0.15 tr. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled 13C–1H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via 13C spin exchange. The efficiencies of these spectral editing techniques range from 60 % for the COO and dynamic selection experiments to 25 % for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.

  17. TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra.

    Zawadzka-Kazimierczuk, Anna; Koźmiński, Wiktor; Billeter, Martin

    2012-09-01

    While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and time-consuming, and the recording of high-dimensional data with high-resolution may be essential. Random sampling of the evolution time space, combined with sparse multidimensional Fourier transform (SMFT), allows for efficient recording of very high dimensional spectra (≥4 dimensions) while maintaining high resolution. However, the nature of this data demands for automation of the assignment process. Here we present the program TSAR (Tool for SMFT-based Assignment of Resonances), which exploits all advantages of SMFT input. Moreover, its flexibility allows to process data from any type of experiments that provide sequential connectivities. The algorithm was tested on several protein samples, including a disordered 81-residue fragment of the δ subunit of RNA polymerase from Bacillus subtilis containing various repetitive sequences. For our test examples, TSAR achieves a high percentage of assigned residues without any erroneous assignments. PMID:22806130

  18. TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra

    Zawadzka-Kazimierczuk, Anna; Kozminski, Wiktor [University of Warsaw, Faculty of Chemistry (Poland); Billeter, Martin, E-mail: martin.billeter@chem.gu.se [University of Gothenburg, Biophysics Group, Department of Chemistry and Molecular Biology (Sweden)

    2012-09-15

    While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and time-consuming, and the recording of high-dimensional data with high-resolution may be essential. Random sampling of the evolution time space, combined with sparse multidimensional Fourier transform (SMFT), allows for efficient recording of very high dimensional spectra ({>=}4 dimensions) while maintaining high resolution. However, the nature of this data demands for automation of the assignment process. Here we present the program TSAR (Tool for SMFT-based Assignment of Resonances), which exploits all advantages of SMFT input. Moreover, its flexibility allows to process data from any type of experiments that provide sequential connectivities. The algorithm was tested on several protein samples, including a disordered 81-residue fragment of the {delta} subunit of RNA polymerase from Bacillus subtilis containing various repetitive sequences. For our test examples, TSAR achieves a high percentage of assigned residues without any erroneous assignments.

  19. Resonance assignment of the NMR spectra of disordered proteins using a multi-objective non-dominated sorting genetic algorithm

    A multi-objective genetic algorithm is introduced to predict the assignment of protein solid-state NMR (SSNMR) spectra with partial resonance overlap and missing peaks due to broad linewidths, molecular motion, and low sensitivity. This non-dominated sorting genetic algorithm II (NSGA-II) aims to identify all possible assignments that are consistent with the spectra and to compare the relative merit of these assignments. Our approach is modeled after the recently introduced Monte-Carlo simulated-annealing (MC/SA) protocol, with the key difference that NSGA-II simultaneously optimizes multiple assignment objectives instead of searching for possible assignments based on a single composite score. The multiple objectives include maximizing the number of consistently assigned peaks between multiple spectra (“good connections”), maximizing the number of used peaks, minimizing the number of inconsistently assigned peaks between spectra (“bad connections”), and minimizing the number of assigned peaks that have no matching peaks in the other spectra (“edges”). Using six SSNMR protein chemical shift datasets with varying levels of imperfection that was introduced by peak deletion, random chemical shift changes, and manual peak picking of spectra with moderately broad linewidths, we show that the NSGA-II algorithm produces a large number of valid and good assignments rapidly. For high-quality chemical shift peak lists, NSGA-II and MC/SA perform similarly well. However, when the peak lists contain many missing peaks that are uncorrelated between different spectra and have chemical shift deviations between spectra, the modified NSGA-II produces a larger number of valid solutions than MC/SA, and is more effective at distinguishing good from mediocre assignments by avoiding the hazard of suboptimal weighting factors for the various objectives. These two advantages, namely diversity and better evaluation, lead to a higher probability of predicting the correct

  20. Empirical Equation Based Chirality (n, m Assignment of Semiconducting Single Wall Carbon Nanotubes from Resonant Raman Scattering Data

    Md Shamsul Arefin

    2012-12-01

    Full Text Available This work presents a technique for the chirality (n, m assignment of semiconducting single wall carbon nanotubes by solving a set of empirical equations of the tight binding model parameters. The empirical equations of the nearest neighbor hopping parameters, relating the term (2n, m with the first and second optical transition energies of the semiconducting single wall carbon nanotubes, are also proposed. They provide almost the same level of accuracy for lower and higher diameter nanotubes. An algorithm is presented to determine the chiral index (n, m of any unknown semiconducting tube by solving these empirical equations using values of radial breathing mode frequency and the first or second optical transition energy from resonant Raman spectroscopy. In this paper, the chirality of 55 semiconducting nanotubes is assigned using the first and second optical transition energies. Unlike the existing methods of chirality assignment, this technique does not require graphical comparison or pattern recognition between existing experimental and theoretical Kataura plot.

  1. Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

    Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to Cα and Cβ separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups

  2. Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

    Mishra, Subrata H.; Frueh, Dominique P., E-mail: dfrueh@jhmi.edu [Johns Hopkins University School of Medicine, Department of Biophysics and Biophysical Chemistry (United States)

    2015-07-15

    Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C{sup α} and C{sup β} separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups.

  3. Nuclear resonance vibrational spectroscopy applied to [Fe(OEP)(NO)] : the vibrational assignments of five-coordinate ferrous heme-nitrosyls and implications for electronic structure.

    Lehnert, N.; Galinato, M. I.; Paulat, F.; Richter-Addo, G. B.; Sturhahn, W.; Xu, N.; Zhao, J. (X-Ray Science Division); (Univ. of Michigan); (Univ. of Oklahoma)

    2010-01-01

    This study presents Nuclear Resonance Vibrational Spectroscopy (NRVS) data on the five-coordinate (5C) ferrous heme-nitrosyl complex [Fe(OEP)(NO)] (1, OEP{sup 2-} = octaethylporphyrinato dianion) and the corresponding {sup 15}N{sup 18}O labeled complex. The obtained spectra identify two isotope sensitive features at 522 and 388 cm{sup -1}, which shift to 508 and 381 cm{sup -1}, respectively, upon isotope labeling. These features are assigned to the Fe-NO stretch v(Fe-NO) and the in-plane Fe-N-O bending mode {delta}{sub ip}(Fe-N-O), the latter has been unambiguously assigned for the first time for 1. The obtained NRVS data were simulated using our quantum chemistry centered normal coordinate analysis (QCC-NCA). Since complex 1 can potentially exist in 12 different conformations involving the FeNO and peripheral ethyl orientations, extended density functional theory (DFT) calculations and QCC-NCA simulations were performed to determine how these conformations affect the NRVS properties of [Fe(OEP)NO]. These results show that the properties and force constants of the FeNO unit are hardly affected by the conformational changes involving the ethyl substituents. On the other hand, the NRVS-active porphyrin-based vibrations around 340-360, 300-320, and 250-270 cm{sup -1} are sensitive to the conformational changes. The spectroscopic changes observed in these regions are due to selective mechanical couplings of one component of Eu-type (in ideal D4h symmetry) porphyrin-based vibrations with the in-plane Fe-N-O bending mode. This leads to the observed variations in Fe(OEP) core mode energies and NRVS intensities without affecting the properties of the FeNO unit. The QCC-NCA simulated NRVS spectra of 1 show excellent agreement with experiment, and indicate that conformer F is likely present in the samples of this complex investigated here. The observed porphyrin-based vibrations in the NRVS spectra of 1 are also assigned based on the QCC-NCA results. The obtained force

  4. 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

  5. 13C-detected NMR experiments for automatic resonance assignment of IDPs and multiple-fixing SMFT processing

    Intrinsically disordered proteins (IDPs) have recently attracted much interest, due to their role in many biological processes, including signaling and regulation mechanisms. High-dimensional 13C direct-detected NMR experiments have proven exceptionally useful in case of IDPs, providing spectra with superior peak dispersion. Here, two such novel experiments recorded with non-uniform sampling are introduced, these are 5D HabCabCO(CA)NCO and 5D HNCO(CA)NCO. Together with the 4D (HACA)CON(CA)NCO, an extension of the previously published 3D experiments (Pantoja-Uceda and Santoro in J Biomol NMR 59:43–50, 2014. doi: 10.1007/s10858-014-9827-1 10.1007/s10858-014-9827-1 ), they form a set allowing for complete and reliable resonance assignment of difficult IDPs. The processing is performed with sparse multidimensional Fourier transform based on the concept of restricting (fixing) some of spectral dimensions to a priori known resonance frequencies. In our study, a multiple-fixing method was developed, that allows easy access to spectral data. The experiments were tested on a resolution-demanding alpha-synuclein sample. Due to superior peak dispersion in high-dimensional spectrum and availability of the sequential connectivities between four consecutive residues, the overwhelming majority of resonances could be assigned automatically using the TSAR program

  6. Multinuclear NMR resonance assignments and the secondary structure of Escherichia coli thioesterase/protease I: A member of a new subclass of lipolytic enzymes

    Escherichia coli thioesterase/protease I is a 183 amino acid protein with a molecular mass of 20500. This protein belongs to a new subclass of lipolytic enzymes of the serine protease superfamily, but with a new GDSLS consensus motif, of which no structure has yet been determined. The protein forms a tetramer at pH values above 6.5 and exists as a monomer at lower pH values. Both monomer and tetramer are catalytically active. From analysis of a set of heteronuclear multidimensional NMR spectra with uniform and specific amino acid labeled protein samples, we have obtained near-complete resonance assignments of the backbone 1H,13 C and 15N nuclei (BMRB databank accession number 4060). The secondary structure of E. coli thioesterase/protease I was further deduced from the consensus chemical shift indices, backbone short- and medium-range NOEs, and amide proton exchange rates. The protein was found to consist of four β-strands and seven α-helices, arranged in alternate order. The four β-strands were shown to form a parallel β-sheet. The topological arrangement of the β-strands of -1x, +2x, +1x appears to resemble that of the core region of the αβ hydrolase superfamily, typically found in common lipases and esterases. However, substantial differences, such as the number of β-strands and the location of the catalytic triad residues, make it difficult to give a definitive classification of the structure of E. coli thioesterase/protease I at present

  7. Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

    Schmidt-Rohr, K.; Fritzsching, K. J.; Liao, S. Y.; Hong Mei, E-mail: mhong@iastate.edu [Iowa State University, Department of Chemistry and Ames Laboratory (United States)

    2012-12-15

    Several techniques for spectral editing of 2D {sup 13}C-{sup 13}C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N-CO peaks through {sup 13}C-{sup 15}N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH{sub 2}) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other {pi}-pulse is shifted from the center of a rotor period t{sub r} by about 0.15 t{sub r}. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled {sup 13}C-{sup 1}H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via {sup 13}C spin exchange. The efficiencies of these spectral editing techniques range from 60 % for the COO and dynamic selection experiments to 25 % for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.

  8. Complete Proton and Carbon Assignment of Triclosan via One- and Two- Dimensional Nuclear Magnetic Resonance Analysis

    Students from an upper-division undergraduate spectroscopy class analyzed one- and two-dimensional 400 MHz NMR spectroscopic data from triclosan in CDCl3. Guided assignment of all proton and carbon signals was completed via 1D proton and carbon, nuclear Overhauser effect (nOe), distortionless enhanc...

  9. High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

    Zawadzka-Kazimierczuk, A.; Kozminski, W.; Šanderová, Hana; Krásný, Libor

    2012-01-01

    Roč. 52, č. 4 (2012), s. 329-337. ISSN 0925-2738 R&D Projects: GA ČR GA204/09/0583 Institutional research plan: CEZ:AV0Z50200510 Keywords : Intrinsically disordered proteins * Non-uniform sampling * Backbone assignment Subject RIV: EE - Microbiology, Virology Impact factor: 2.845, year: 2012

  10. Inhibition of alanine racemase by alanine phosphonate: detection of an imine linkage to pyridoxal 5'-phosphate in the enzyme-inhibitor complex by solid-state 15N nuclear magnetic resonance

    Inhibition of alanine racemase from the Gram-positive bacterium Bacillus stearothermophilus by (1-aminoethyl)phosphonic acid (Ala-P) proceeds via a two-step reaction pathway in which reactivation occurs very slowly. In order to determine the mechanism of inhibition, the authors have recorded low-temperature, solid-state 15N NMR spectra from microcrystals of the [15N]Ala-P-enzyme complex, together with spectra of a series of model compounds that provide the requisite database for the interpretation of the 15N chemical shifts. Proton-decoupled spectra of the microcrystals exhibit a line at ∼ 150 ppm, which conclusively demonstrates the presence of a protonated Ala-P-PLP aldimine and thus clarifies the structure of the enzyme-inhibitor complex. They also report the pH dependence of Ala-P binding to alanine racemase

  11. Complete assignment of lysine resonances in 1H NMR spectra of proteins as probes of surface structure and dynamics

    A strategy is presented for complete identification of 1H spin systems of lysine residues using sophisticated 2D NMR experiments. Relayed and remote connectivities within each spin system are determined for spin subsystems based at the backbone amide and Cε proton resonances. When complete spin system identification is combined with sequence-specific assignment, protein surface structure and dynamics can be probed in a site-specific manner. The interaction between the five lysine residues of French bean plastocyanin and a model redox partner Cr(CN)63- has been examined using this approach. 12 refs.; 3 figs.; 1 table

  12. Assignment of resonances in dissociative recombination of HD+ ions: high-resolution measurements compared with accurate computations

    Tamo, F O Waffeu; Motapon, O; Altevogt, S; Andrianarijaona, V M; Grieser, M; Lammich, L; Lestinsky, M; Motsch, M; Nevo, I; Novotny, S; Orlov, D A; Pedersen, H B; Schwalm, D; Sprenger, F; Urbain, X; Weigel, U; Wolf, A; Schneider, I F

    2011-01-01

    The collision-energy resolved rate coefficient for dissociative recombination of HD+ ions in the vibrational ground state is measured using the photocathode electron target at the heavy-ion storage ring TSR. Rydberg resonances associated with ro-vibrational excitation of the HD+ core are scanned as a function of the electron collision energy with an instrumental broadening below 1 meV in the low-energy limit. The measurement is compared to calculations using multichannel quantum defect theory, accounting for rotational structure and interactions and considering the six lowest rotational energy levels as initial ionic states. Using thermal equilibrium level populations at 300 K to approximate the experimental conditions, close correspondence between calculated and measured structures is found up to the first vibrational excitation threshold of the cations near 0.24 eV. Detailed assignments, including naturally broadened and overlapping Rydberg resonances, are performed for all structures up to 0.024 eV. Resona...

  13. Deuterium-labeling method for the assignment of histidine nuclear magentic resonance peaks of proteins

    A tritium labelling method involving differential tritium exchange at the C-2 H position of histidines and 1H NMR of differentially deuterated proteins can be a general method for the assignment of the histidine NMR peaks. In the present report this method is modified by replacing the tritium with deuterium, which eliminates ambiguities arising from the tritium isotope effect. In the deuterium labelling method, differentially deuterated proteins are cleaved by trypsin into smaller peptides each containing a single histidine residue, which are separated by chromatography. The method was applied to the Bence Hones dimer Ak which contains two histidine residues in the constant domain of each of the light chains. The decay of the NMR peaks with time allowed the assignment of one peak to His189 and the other to His198

  14. Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis

    Bumba, Ladislav; Sviridova, E.; Kutá-Smatanová, Ivana; Řezáčová, Pavlína; Veverka, Václav

    2014-01-01

    Roč. 8, č. 1 (2014), s. 53-55. ISSN 1874-2718 R&D Projects: GA ČR(CZ) GAP207/11/0717; GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:61388971 ; RVO:67179843 Keywords : Neisseria meningitidis * FrpC * FrpD * backbone assignments * NMR * iron-regulated protein Subject RIV: CE - Biochemistry Impact factor: 0.760, year: 2014

  15. Backbone resonance assignments of human cytosolic dNT-1 nucleotidase

    Hnízda, Aleš; Skleničková, Radka; Pachl, Petr; Fábry, Milan; Tošner, Z.; Brynda, Jiří; Veverka, Václav

    2014-01-01

    Roč. 8, č. 2 (2014), s. 425-428. ISSN 1874-2718 R&D Projects: GA MŠk(CZ) LK11205; GA ČR GA203/09/0820 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : 5 '-nucleotidase * haloacid dehalogenase superfamily * backbone assignments * NMR * perdeuterated protein * dimer * pyrimidine nucleotides Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J) Impact factor: 0.760, year: 2014

  16. Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

    The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonance of the heme and ligand τ nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine π nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine π NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine causes gross conformational change near the heme center. There are multiple conformations at high pH, as judged by saturation-transfer experiments. The N-terminus of the ferricytochrome has a pKa of 8.6. In contrast to its partially restricted mobility in the reduced state, it is found to be very mobile, reflecting a looser structure of the ferricytochrome

  17. Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

    Puthenpurackal Narayanan, Sunilkumar; Maeno, Akihiro; Wada, Yuji; Tate, Shin-Ichi; Akasaka, Kazuyuki

    2016-04-01

    Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(β) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35 °C, and then evaluated the H(N), N(H), C(α) and C(β) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain. PMID:26482924

  18. Dosimetric evaluation of synthetic CT relative to bulk density assignment-based magnetic resonance-only approaches for prostate radiotherapy

    Magnetic resonance imaging (MRI) has been incorporated as an adjunct to CT to take advantage of its excellent soft tissue contrast for contouring. MR-only treatment planning approaches have been developed to avoid errors introduced during the MR-CT registration process. The purpose of this study is to evaluate calculated dose distributions after incorporating a novel synthetic CT (synCT) derived from magnetic resonance simulation images into prostate cancer treatment planning and to compare dose distributions calculated using three previously published MR-only treatment planning methodologies. An IRB-approved retrospective study evaluated 15 prostate cancer patients that underwent IMRT (n = 11) or arc therapy (n = 4) to a total dose of 70.2-79.2 Gy. Original treatment plans were derived from CT simulation images (CT-SIM). T1-weighted, T2-weighted, and balanced turbo field echo images were acquired on a 1.0 T high field open MR simulator with patients immobilized in treatment position. Four MR-derived images were studied: bulk density assignment (10 HU) to water (MRW), bulk density assignments to water and bone with pelvic bone values derived either from literature (491 HU, MRW+B491) or from CT-SIM population average bone values (300 HU, MRW+B300), and synCTs. Plans were recalculated using fixed monitor units, plan dosimetry was evaluated, and local dose differences were characterized using gamma analysis (1 %/1 mm dose difference/distance to agreement). While synCT provided closest agreement to CT-SIM for D95, D99, and mean dose (<0.7 Gy (1 %)) compared to MRW, MRW+B491, and MRW+B300, pairwise comparisons showed differences were not significant (p < 0.05). Significant improvements were observed for synCT in the bladder, but not for rectum or penile bulb. SynCT gamma analysis pass rates (97.2 %) evaluated at 1 %/1 mm exceeded those from MRW (94.7 %), MRW+B300 (94.0 %), or MRW+B491 (90.4 %). One subject’s synCT gamma (1 %/1 mm) results (89.9 %) were lower than MRW

  19. 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)

  20. Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment

    13C- and 2H-labeled retinal derivatives have been used to assign normal modes in the 1100-1300-cm-1 fingerprint region of the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. On the basis of the 13C shifts, C8-C9 stretching character is assigned at 1217 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1214 cm-1 in bathorhodopsin. C10-C11 stretching character is localized at 1098 cm-1 in rhodopsin, at 1154 cm-1 in isorhodopsin, and at 1166 cm-1 in bathorhodopsin. C14-C15 stretching character is found at 1190 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1210 cm-1 in bathorhodopsin. C12-C13 stretching character is much more delocalized, but the characteristic coupling with the C14H rock allows the authors to assign the C12-C13 stretch at ∼1240 cm-1 in rhodopsin, isorhodopsin, and bathorhodopsin. The insensitivity of the C14-C15 stretching mode to N-deuteriation in all three pigments demonstrates that each contains a trans (anti) protonated Schiff base bond. The relatively high frequency of the C10-C11 mode of bathorhodopsin demonstrates that bathorhodopsin is s-trans about the C10-C11 single bond. This provides strong evidence against the model of bathorhodopsin proposed by Liu and Asato, which suggests a C10-C11 s-cis structure. Comparison of the fingerprint modes of rhodopsin with those of the 11-cis-retinal protonated Schiff base in methanol shows that the frequencies of the C-C stretching modes are largely unperturbed by protein binding. The implications of these observations for the mechanism of wavelength regulation in visual pigments and energy storage in bathorhodopsin are discussed

  1. Differentiation of Histidine Tautomeric States using 15N Selectively Filtered 13C Solid-State NMR Spectroscopy

    Miao, Yimin; Cross, Timothy A.; Fu, Riqiang

    2014-01-01

    The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional 15N selectively filtered 13C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all 13C resonances of the individual imidazole rings in a mixture of tautomeric states. When 15N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the 13C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of 13C,15N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture. PMID:25026459

  2. Resonance assignment of an engineered amino-terminal domain of a major ampullate spider silk with neutralized charge cluster.

    Schaal, Daniel; Bauer, Joschka; Schweimer, Kristian; Scheibel, Thomas; Rösch, Paul; Schwarzinger, Stephan

    2016-04-01

    Spider dragline fibers are predominantly made out of the major ampullate spidroins (MaSp) 1 and 2. The assembly of dissolved spidroin into a stable fiber is highly controlled for example by dimerization of its amino-terminal domain (NRN) upon acidification, as well as removal of sodium chloride along the spinning duct. Clustered residues D39, E76 and E81 are the most highly conserved residues of the five-helix bundle, and they are hypothesized to be key residues for switching between a monomeric and a dimeric conformation. Simultaneous replacement of these residues by their non-titratable analogues results in variant D39N/E76Q/E81Q, which is supposed to fold into an intermediate conformation between that of the monomeric and the dimeric state at neutral pH. Here we report the resonance assignment of Latrodectus hesperus NRN variant D39N/E76Q/E81Q at pH 7.2 obtained by high-resolution triple resonance NMR spectroscopy. PMID:26892754

  3. Assignment of hyperfine shifted haem methyl carbon resonances in paramagnetic low-spin met-cyano complex of sperm whale myoglobin

    The hyperfine shifted resonances arising from all four individual haem carbons of the paramagnetic low-spin met-cyano complex of sperm whale myoglobin have been clearly identified and assigned for the first time with the aid of 1H-13C heteronuclear chemical shift correlated spectroscopy. Alteration of the in-plane symmetry of the electronic structure of haem induced by the ligation of proximal histidyl imidazole spreads the haem carbon resonances to 32 ppm at 220C, indicating the sensitivity of those resonances to the haem electronic/molecular structure. Those resonances are potentially powerful probes in characterizing the nature of haem electronic structure. 25 refs.; 2 figs.; 1 table

  4. 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.

  5. 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

  6. 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

  7. Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein

    Doležal, Michal; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2015-01-01

    Roč. 9, č. 2 (2015), s. 229-233. ISSN 1874-2718 Institutional support: RVO:61388963 Keywords : isotopic labeling * matrix protein * M-PMV * myristoylation * resonance assignment * reverse labeling Subject RIV: CE - Biochemistry Impact factor: 0.760, year: 2014

  8. Sequence-specific Assignment of 1H-NMR Resonance and Determination of the Secondary Structure of Jingzhaotoxin-Ⅰ

    Xiong-Zhi ZENG; Qi ZHU; Song-Ping LIANG

    2005-01-01

    Jingzhaotoxin-Ⅰ (JZTX-Ⅰ) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3.The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-Ⅰ were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by dαN,dαδ, dβN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-Ⅰ. Furthermore, the secondary structure of JZTX-Ⅰ was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-Ⅰ are similar to those of huwentoxin-Ⅰ (HWTX-Ⅰ) and hainantoxin-Ⅳ (HNTX-Ⅳ), whose structures in solution have previously been reported.

  9. Carbonyl 13C NMR spectrum of basin pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

    The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering

  10. Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods

    Penzel, Susanne; Smith, Albert A.; Agarwal, Vipin; Hunkeler, Andreas [ETH Zürich, Physical Chemistry (Switzerland); Org, Mai-Liis; Samoson, Ago, E-mail: ago.samoson@ttu.ee [Tallinn University of Technology, NMR Instituut, Tartu Teadus, Tehnomeedikum (Estonia); Böckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Université de Lyon 1, Institut de Biologie et Chimie des Protéines (France); Ernst, Matthias, E-mail: maer@ethz.ch; Meier, Beat H., E-mail: beme@ethz.ch [ETH Zürich, Physical Chemistry (Switzerland)

    2015-10-15

    We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T{sub 2}′ times and a site-specific comparison of T{sub 2}′ at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96 %.

  11. 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.

  12. 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)

  13. ¹H, ¹³C and ¹⁵N resonance assignment of the soluble form of the lipid-modified Azurin from Neisseria gonorrhoeae.

    Nóbrega, Cláudia S; Matzapetakis, Manolis; Pauleta, Sofia R

    2013-10-01

    Lipid-modified azurin (Laz) from Neisseria gonorrhoeae is a type 1 copper protein proposed to be the electron donor to several enzymes involved in the resistance mechanism to reactive oxygen and nitrogen species. Here we report the backbone and side-chain resonance assignment of Laz in the reduced form, which has been complete at 97%. The predicted secondary structure indicates that this protein belongs to the azurin subfamily of type 1 copper proteins. PMID:23070845

  14. Two- and three-dimensional sup 1 H NMR studies of a wheat phospholipid transfer protein: Sequential resonance assignments and secondary structure

    Simorre, J.P.; Caille, A. (Centre National de la Recherche Scientifique, Orleans (France)); Marion, D. (Laboratoire de Resonance Magnetique en Biologie et Medecine, Grenoble (France)); Marion, D. (INRA, Nantes (France)); Ptak, M. (Centre National de la Recherche Scientifique, Orleans (France) Univ. d' Orleans (France))

    1991-12-10

    Two- and three-dimensional {sup 1}H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy prove to be a very efficient method for resonance assignments of protein {sup 1}H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by {sup 3}J{sub {alpha}NH} coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.

  15. 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.

  16. 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.

  17. 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.)

  18. 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)

  19. Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

    A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKa's for the six histidines in this enzyme. The pKa's of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg

  20. 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...

  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. 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

  3. Amino-acid selective experiments on uniformly 13C and 15N labeled proteins by MAS NMR: Filtering of lysines and arginines

    Jehle, Stefan; Rehbein, Kristina; Diehl, Anne; van Rossum, Barth-Jan

    2006-12-01

    Amino-acid selective magic-angle spinning (MAS) NMR experiments can aid the assignment of ambiguous cross-peaks in crowded spectra of solid proteins. In particular for larger proteins, data analysis can be hindered by severe resonance overlap. In such cases, filtering techniques may provide a good alternative to site-specific spin-labeling to obtain unambiguous assignments that can serve as starting points in the assignment procedure. In this paper we present a simple pulse sequence that allows selective excitation of arginine and lysine residues. To achieve this, we make use of a combination of specific cross-polarization for selective excitation [M. Baldus, A.T. Petkova, J. Herzfeld, R.G. Griffin, Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems, Mol. Phys. 95 (1998) 1197-1207.] and spin diffusion for transfer along the amino-acid side-chain. The selectivity of the filter is demonstrated with the excitation of lysine and arginine side-chain resonances in a uniformly 13C and 15N labeled protein preparation of the α-spectrin SH3 domain. It is shown that the filter can be applied as a building block in a 13C- 13C lysine-only correlation experiment.

  4. A tracked approach for automated NMR assignments in proteins (TATAPRO)

    A novel automated approach for the sequence specific NMR assignments of 1HN, 13Cα, 13Cβ, 13C'/1Hα and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of 13Cα, 13Cβ and 13C'/1Hα. The information derived from such correlations is used to create a 'masterlist' consisting of all possible sets of 1HNi, 15Ni, 13Cαi, 13Cβi, 13C'i/1Hαi, 13Cαi-1, 13Cβi-1 and 13C'i-1/ 1Hαi-1 chemical shifts. On the basis of an extensive statistical analysis of 13Cα and 13Cβ chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the masterlist and also to translate the protein primary sequence into an array called ppsarray. The program then uses the masterlist to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so, each assigned residue is tracked in an array called assigarray, with the two-digit code assigned earlier. The assigarray is then mapped onto the ppsarray for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18-42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the masterlist

  5. 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

  6. Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

    Truhlar, Stephanie M E; Cervantes, Carla F; Torpey, Justin W;

    2008-01-01

    analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used......Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label...... during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by...

  7. 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

  8. 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.

  9. 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)

  10. 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)

  11. 4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy.

    Stivers, J T; Abeygunawardana, C; Whitman, C. P.; Mildvan, A. S.

    1996-01-01

    4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta 1, residu...

  12. Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: Assignment of aromatic resonances with application to protein folding, structure, and dynamics

    The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25 → Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constrains in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures

  13. Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: Assignment of aromatic resonances with application to protein folding, structure, and dynamics

    Weiss, M.A.; Shoelson, S.E. (Harvard Medical School, Boston, MA (USA) Massachusetts General Hospital, Boston (USA)); Nguyen, D.T.; O' Shea, E.; Karplus, M. (Harvard Univ., Cambridge, MA (USA)); Khait, I.; Neuringer, L.J. (Massachusetts Institute of Technology, Cambridge (USA)); Inouye, K. (Shionogi and Co., Ltd., Osaka (Japan)); Frank, B.H.; Beckage, M. (Eli Lilly and Co., Indianapolis, IN (USA))

    1989-12-12

    The aromatic {sup 1}H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25 {yields} Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constrains in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.

  14. 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)

  15. Preparation of 15N labelled protein sample by gene engineering technology

    Using the advanced multi-dimension heteronuclear pulses and isotope labelled protein technique, nuclear magnetic resonance spectroscopy has become an important tool in analysis of the solution conformation of protein. On the basis of the high level expression of a protein-trichosanthin in recombinant E.coli using DNA, 15N was used to label the protein, the 15N labelled trichosanthin was obtained by affinity chromatography on Ni-NTA agarose. Terminating pregnant effect in mice showed that this recombinant protein had the same activity as natural trichosanthin. A 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum was obtained from an AM-500 NMR spectrometer, demonstrating that this method is suitable in preparing labelled protein sample for NMR

  16. Renal ischemia and reperfusion assessment with three-dimensional hyperpolarized (13) C,(15) N2-urea

    Nielsen, Per Mose; Szocska Hansen, Esben Søvsø; Nørlinger, Thomas Stokholm;

    2016-01-01

    . METHODS: Hyperpolarized three-dimensional balanced steady-state (13) C magnetic resonance imaging (MRI) experiments alongside kidney function parameters and quantitative polymerase chain reaction measurements were performed in rats subjected to unilateral renal ischemia for 60-minute and 24-hour......,(15) N2 urea MRI can be used to successfully detect changes in the intrarenal urea gradient post-IRI, thereby enabling in vivo monitoring of the intrarenal functional status in the rat kidney. Magn Reson Med, 2016. © 2016 International Society for Magnetic Resonance in Medicine....

  17. 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

  18. 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)

  19. 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

  20. 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)

  1. 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)

  2. Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

    Truhlar, Stephanie M.E.; Cervantes, Carla F.; Torpey, Justin W.; Kjaergaard, Magnus; Komives, Elizabeth A.

    2008-01-01

    Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific 15N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific 15N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The 15N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the 15N-amino acid prevents the scrambling of the 15N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the “extra” 15N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples. PMID:18567787

  3. 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)

  4. Sequence-Specific Assignment and Secondary Structure of the Catalytic Domain of Protein from Ubiquitination Pathway

    Ubiquitination is a post-translational protein modification which plays an important role in a wide variety of cellular processes including cell cycle, DNA repair and cell apoptosis. It is well known, that the ubiquitination requires sequential activity of three enzymes with different functions: activation, conjugation and ligation. Unfortunately, the three-dimensional structures of all three proteins responsible for these processes are not available at present and the process of proteins ubiquitination still is not understood in detail. In our communication, we present first, preliminary NMR data for the sequence-specific assignments for 112 amino acid residues long domain of one of the proteins from the ubiquitination pathway. The NMR samples were prepared by dissolving 1 mm either 15N-labeled or 15N, 13C-double labeled protein in 90%/10% H2O/D2O, 50 mm TRIS buffer, and 50 mm NaCl. The ph was adjusted to 6.5 (uncorrected value). All NMR measurements were performed on the Varian Unity+ 500 NMR spectrometer (11.7 T) equipped with three channels, Performa II PFG unit and 5 mm 1H, 13C, 15N-triple resonance pro behead. The 1H, 15N, and 13C backbone resonances were assigned by standard methods using 3D heteronuclear HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, HNCO, (HCA)CO(CA)NH NMR spectra collected at 303 K. The aliphatic 1H and 13C resonances were assigned on the basis of C(CO)NH, HBHA(CO)NH, and H(CO)NH experiments. After finishing of assignment procedure, solution of secondary structure in studied protein has been performed. The exact position of the α-helices and β-strands were solved on base analysis of cross-peaks between HN and Hα protons in 3D 15N-edited NOESY-HSQC spectrum, 3JNHα coupling constants evaluated from 3D HNHA experiment, and chemical shifts of backbone nuclei (TALOS software). Obtained results will be used in future for solution of three-dimensional structure of catalytic domain with high resolution by means NMR methods. (author)

  5. 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)

  6. A tracked approach for automated NMR assignments in proteins (TATAPRO)

    Atreya, H.S.; Sahu, S.C.; Chary, K.V.R.; Govil, Girjesh [Tata Institute of Fundamental Research, Department of Chemical Sciences (India)

    2000-06-15

    A novel automated approach for the sequence specific NMR assignments of {sup 1}H{sup N}, {sup 13}C{sup {alpha}}, {sup 13}C{sup {beta}}, {sup 13}C'/{sup 1}H{sup {alpha}} and {sup 15}N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate {sup 1}H{sup N} and {sup 15}N chemical shifts with those of {sup 13}C{sup {alpha}}, {sup 13}C{sup {beta}} and {sup 13}C'/{sup 1}H{sup {alpha}}. The information derived from such correlations is used to create a 'master{sub l}ist' consisting of all possible sets of {sup 1}H{sup N}{sub i}, {sup 15}N{sub i}, {sup 13}C{sup {alpha}}{sub i}, {sup 13}C{sup {beta}}{sub i}, {sup 13}C'{sub i}/{sup 1}H{sup {alpha}}{sub i}, {sup 13}C{sup {alpha}}{sub i-1}, {sup 13}C{sup {beta}}{sub i-1} and {sup 13}C'{sub i-1}/ {sup 1}H{sup {alpha}}{sub i-1} chemical shifts. On the basis of an extensive statistical analysis of {sup 13}C{sup {alpha}} and {sup 13}C{sup {beta}} chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master{sub l}ist and also to translate the protein primary sequence into an array called pps{sub a}rray. The program then uses the master{sub l}ist to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so, each assigned residue is tracked in an array called assig{sub a}rray, with the two-digit code assigned earlier. The assig{sub a}rray is then mapped onto the pps{sub a}rray for sequence specific resonance assignment. The program has been tested using

  7. Pentacyclic triterpenoids of Mentha villosa: structural identification and {sup 1}H and {sup 13}C resonance assignments; Triterpenoides pentaciclicos de Mentha villosa: identificacao estrutural e atribuicao dos deslocamentos quimicos dos atomos de hidrogenio e carbono

    Monte, Francisco J. Queiroz; Oliveira, Eliete F. de [Ceara Univ., Fortaleza, CE (Brazil). Dept. de Quimica Organica e Inorganica; Braz Filho, Raimundo [Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ (Brazil). Setor de Quimica de Produtos Naturais

    2001-08-01

    The structures of seven oleanene and ursene triterpenoids (1-7) isolated from aerial parts of Mentha villosa were identified. In addition, the complete {sup 1} H and {sup 13} C resonance assignments of these triterpenoids were accomplished using 1D and 2D NMR spectroscopic experiments. (author)

  8. 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

  9. Nature of organic carbon and nitrogen in physically protected organic matter of some Australian soils as revealed by solid-state 13 C and 15 N NMR spectroscopy

    The 13C and 15N nuclear magnetic resonance (NMR) spectroscopy was applied for characterising the chemical nature of the remaining organic fraction. The 13C NMR spectroscopic comparison of the residues after UV photo-oxidation and the untreated bulk soils revealed a considerable increase in condensed aromatic structures in the residues for 4 of the 5 soils. This behaviour was recently shown to be typical for char-containing soils. In the sample where no char was detectable by NMR spectroscopy, the physically protected carbon consisted of functional groups similar to those observed for the organic matter of the bulk sample, although their relative proportions were altered. The solid-state 15N NMR spectrum from this sample revealed that some peptide structures were able to resist UV photo-oxidation, probably physically protected within the core of micro aggregates. Heterocyclic aromatic nitrogen was not detected in this spectrum, but pyrrolic nitrogen was found to comprise a major fraction of the residues after photo-oxidation of the <53 μm containing soils. Acid hydrolysis of these samples confirmed that some peptide-like material was still present. The identification of a considerable amount of aromatic carbon and nitrogen, assignable to charred material in 4 of the 5 investigated soils, supports previous observations that char largely comprises the inert or passive organic matter pool of many Australian soils. The influence of such material on the carbon and nitrogen dynamics in such soils, however, requires further research. Copyright (2000) CSIRO Australia

  10. The $^{15}$N($\\bm\\alpha$,$\\bm\\gamma$)$^{19}$F reaction and nucleosynthesis of $^{19}$F

    Wilmes, S.; Wilmes, V.; Staudt, G.; Mohr, P; Hammer, J. W.

    2002-01-01

    Several resonances in the $^{15}$N($\\alpha$,$\\gamma$)$^{19}$F reaction have been investigated in the energy range between 0.6 MeV and 2.7 MeV. Resonance strengths and branching ratios have been determined. High sensitivity could be obtained by the combination of the {\\sc{dynamitron}} high current accelerator, the windowless gas target system {\\sc{rhinoceros}}, and actively shielded germanium detectors. Two levels of $^{19}$F could be observed for the first time in the ($\\alpha$,$\\gamma$) chan...

  11. 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

  12. 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)

  13. 15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate

    The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5-15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex. The 15N spectra of the binary enzyme-folate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7-proton of bound folate, located by two-dimensional exchange spectroscopy. 15 refs.; 3 figs.; 1 table

  14. 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)

  15. 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)

  16. 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)

  17. Polynomial Assignments

    Guillemin, Victor; Sabatini, Silvia; Zara, Catalin

    2013-01-01

    The concept of assignments was introduced in [GGK99] as a method for extracting geometric information about group actions on manifolds from combinatorial data encoded in the infinitesimal orbit-type stratification. In this paper we will answer in the affirmative a question posed in [GGK99] by showing that the equivariant cohomology ring of $M$ is to a large extent determined by this data.

  18. Resonance-enhanced multiphoton ionization (REMPI) spectroscopy of bromobenzene and its perdeuterated isotopologue: Assignment of the vibrations of the S(0), S(1), and D(0)(+) states of bromobenzene and the S(0) and D(0)(+) states of iodobenzene.

    Andrejeva, Anna; Tuttle, William D; Harris, Joe P; Wright, Timothy G

    2015-12-28

    We report vibrationally resolved spectra of the S1←S0 transition of bromobenzene using resonance-enhanced multiphoton ionization spectroscopy. We study bromobenzene-h5 as well as its perdeuterated isotopologue, bromobenzene-d5. The form of the vibrational modes between the isotopologues and also between the S0 and S1 electronic states is discussed for each species, allowing assignment of the bands to be achieved and the activity between states and isotopologues to be established. Vibrational bands are assigned utilizing quantum chemical calculations, previous experimental results, and isotopic shifts. Previous work and assignments of the S1 spectra are discussed. Additionally, the vibrations in the ground state cation, D0 (+), are considered, since these have also been used by previous workers in assigning the excited neutral state spectra. We also examine the vibrations of iodobenzene in the S0 and D0 (+) states and comment on the previous assignments of these. In summary, we have been able to assign the corresponding vibrations across the whole monohalobenzene series of molecules, in the S0, S1, and D0 (+) states, gaining insight into vibrational activity and vibrational couplings. PMID:26723684

  19. Resonance-enhanced multiphoton ionization (REMPI) spectroscopy of bromobenzene and its perdeuterated isotopologue: Assignment of the vibrations of the S0, S1, and D0+ states of bromobenzene and the S0 and D0+ states of iodobenzene

    We report vibrationally resolved spectra of the S1←S0 transition of bromobenzene using resonance-enhanced multiphoton ionization spectroscopy. We study bromobenzene-h5 as well as its perdeuterated isotopologue, bromobenzene-d5. The form of the vibrational modes between the isotopologues and also between the S0 and S1 electronic states is discussed for each species, allowing assignment of the bands to be achieved and the activity between states and isotopologues to be established. Vibrational bands are assigned utilizing quantum chemical calculations, previous experimental results, and isotopic shifts. Previous work and assignments of the S1 spectra are discussed. Additionally, the vibrations in the ground state cation, D0+, are considered, since these have also been used by previous workers in assigning the excited neutral state spectra. We also examine the vibrations of iodobenzene in the S0 and D0+ states and comment on the previous assignments of these. In summary, we have been able to assign the corresponding vibrations across the whole monohalobenzene series of molecules, in the S0, S1, and D0+ states, gaining insight into vibrational activity and vibrational couplings

  20. 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)

  1. Spin and parity determinations of excited 15N based on polarized and unpolarized 12C(7Li, α)15N reaction data at E lab = 34 MeV

    From an experiment conducted at the Florida State University Accelerator Laboratory with a 34 MeV polarized 7Li beam bombarding a 12C target, we have obtained angular distributions and analyzing powers for states of 15N up to 20 MeV in excitation energy. This study not only offers the possibility to assign spin and parity to several states in 15N, but also serves to obtain nuclear potential parameters used in Distorted Wave Born (DWBA) and Coupled Channel Born (CCBA) Approximations to generate theoretical angular distributions and vector analyzing powers that give the best description of the experimental data. Under the assumption that the reaction mechanism is a three nucleon transfer, the determination of shell model nucleonic configurations and spectroscopic factors is possible for the 15N states studied

  2. Carbon-13 nuclear magnetic resonance spectroscopy of [1-13C] enriched monosaccharides. Signal assignments and orientational dependence of geminal and vicinal carbon--carbon and carbon--hydrogen spin--spin coupling constants

    Early assignments of the 13C resonances in the natural abundance 13C NMR spectra of monosaccharides have been reevaluated in light of recent coupling data from the spectra of 13C-1 labeled sugars. The technique of specific 13C enrichment not only identifies the labeled carbon unambiguously but can be used to assign more remote carbon resonances due to scalar carbon-carbon coupling. The pattern of carbon-carbon coupling observed in all of the sugars thus far studied is remarkably constant. In addition to the large (approximately 46 Hz) one-bond coupling between C-1 and C-2, C-3 exhibits a coupling to C-1 only in the β anomer (approximately 4 Hz) while C-5 is coupled to C-1 only in the α anomer (approximately 2 Hz). In addition, C-6 is coupled to C-1 in both anomers and C-4 shows no evidence of coupling to C-1 in any of the sugars examined. These couplings have been used to reassign several resonances and the original assignments are discussed in terms of the predictive rules used for resonance assignments in carbohydrates. The vicinal couplings of C-6 and C-4 to C-1 appear to obey a Karplus-type relationship. The geminal 2J/sub CCC/ and 2J/sub COC/ couplings are discussed in terms of a dihedral angle dependence where the angle is defined by the relative orientations of C-3 or C-5 and the electronegative oxygen substituents on C-1. Additional data on 2J/sub CCH/ couplings involving C-1 and H-2 are also readily obtained with the C-1 labeled sugars

  3. 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)

  4. 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.

  5. 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)

  6. 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.)

  7. 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 ...

  8. Binary reaction channels in the 12C+19F and 16O+15N nuclear collisions

    The 19F on 12C and 15N on 16O reactions are studied not only in order to search for resonances but furthermore to perform a comparative study of binary reaction channels in two collisions leading to the same excitation energies of the composite system. The main feature of the experimental procedure is an exclusive detection of the two fragments in the exit channel using the kinematical coincidence method. Angular distributions and excitation functions of the main binary channels are presented and discussed

  9. Two-dimensional 1H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain

    Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includes two and a half turns of α-helix running from residues 21 to 30 and a three-stranded antiparallel β-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the α-helix to an external strand of the β-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction

  10. 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

  11. Double resonance capacitance spectroscopy (DORCAS): A new experimental technique for assignment of X-ray absorption peaks to surface sites of semiconductor

    Ishii, M

    2003-01-01

    As a new microspectroscopy for semiconductor surface analysis using an X-ray beam, double resonance capacitance spectroscopy (DORCAS) is proposed. For a microscopic X-ray absorption measurement, a local capacitance change owing to X-ray induced emission of localized electrons is detected by a microprobe. The applied bias voltage V sub b dependence of the capacitance also provides information on the surface density of state. The resonance of the Fermi energy with a surface level by V sub b control makes possible the selection of the observable surface site in the X-ray absorption measurements, i.e. site-specific spectroscopy. The double resonance of the surface site selection (V sub b resonance) and the resonant X-ray absorption of the selected site (photon energy h nu resonance) enhances the capacitance signal. The DORCAS measurement of the GaAs surface shows correlation peaks at h nu=10.402 keV and V sub b =-0.4 V and h nu=10.429 keV and V sub b =+0.1 V, indicating that these resonant X-ray absorption peaks ...

  12. Millimeter-wave optical double resonance schemes for rapid assignment of perturbed spectra, with applications to the C{sup ~} {sup 1}B{sub 2} state of SO{sub 2}

    Park, G. Barratt, E-mail: barratt@mit.edu, E-mail: barratt.park@gmail.com; Womack, Caroline C.; Jiang, Jun; Field, Robert W., E-mail: rwfield@mit.edu [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Whitehill, Andrew R.; Ono, Shuhei [Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States)

    2015-04-14

    Millimeter-wave detected, millimeter-wave optical double resonance (mmODR) spectroscopy is a powerful tool for the analysis of dense, complicated regions in the optical spectra of small molecules. The availability of cavity-free microwave and millimeter wave spectrometers with frequency-agile generation and detection of radiation (required for chirped-pulse Fourier-transform spectroscopy) opens up new schemes for double resonance experiments. We demonstrate a multiplexed population labeling scheme for rapid acquisition of double resonance spectra, probing multiple rotational transitions simultaneously. We also demonstrate a millimeter-wave implementation of the coherence-converted population transfer scheme for background-free mmODR, which provides a ∼10-fold sensitivity improvement over the population labeling scheme. We analyze perturbations in the C{sup ~} state of SO{sub 2}, and we rotationally assign a b{sub 2} vibrational level at 45 328 cm{sup −1} that borrows intensity via a c-axis Coriolis interaction. We also demonstrate the effectiveness of our multiplexed mmODR scheme for rapid acquisition and assignment of three predissociated vibrational levels of the C{sup ~} state of SO{sub 2} between 46 800 and 47 650 cm{sup −1}.

  13. Isotope effects and spectroscopic assignments in the non-dissociative photoionization spectrum of N{sub 2}

    Randazzo, John B.; Croteau, Philip [Department of Chemistry, University of California, Berkeley, California 94720 (United States); Kostko, Oleg; Ahmed, Musahid [Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (United States); Boering, Kristie A. [Department of Chemistry, University of California, Berkeley, California 94720 (United States); Department of Earth and Planetary Science, University of California, Berkeley, California 94720 (United States)

    2014-05-21

    Photoionization efficiency spectra of {sup 14}N{sub 2}, {sup 15}N{sup 14}N, and {sup 15}N{sub 2} from 15.5 to 18.9 eV were measured using synchrotron radiation at the Advanced Light Source at Lawrence Berkeley National Laboratory with a resolution of 6 meV, and significant changes in peak energies and intensities upon isotopic substitution were observed. Previously, we reported the isotope shifts and their applications to Titan's atmosphere. Here, we report more extensive experimental details and tabulate the isotope shifts of many transitions in the N{sub 2} spectrum, including those for {sup 15}N{sup 14}N, which have not been previously reported. The isotope shifts are used to address several long-standing ambiguities in spectral peak assignments just above the ionization threshold of N{sub 2}. The feature at 15.677 eV (the so-called second “cathedral” peak) is of particular interest in this respect. The measured isotope shifts for this peak relative to {sup 14}N{sub 2} are 0.015 ± 0.001 eV for {sup 15}N{sub 2} and 0.008 ± 0.001 eV for {sup 15}N{sup 14}N, which match most closely with the isotope shifts predicted for transitions to the (A {sup 2}Π{sub u} v{sup ′} = 2)4sσ{sub g} {sup 1}Π{sub u} state using Herzberg equations for the isotopic differences in harmonic oscillator energy levels plus the first anharmonic correction of 0.0143 eV for {sup 15}N{sub 2} and 0.0071 eV for {sup 15}N{sup 14}N. More generally, the isotope shifts measured for both {sup 15}N{sub 2} and {sup 15}N{sup 14}N relative to {sup 14}N{sub 2} provide new benchmarks for theoretical calculations of interferences between direct and indirect autoionization states which can interact to produce intricate resonant structures in molecular photoionization spectra in regions near ionization thresholds.

  14. Fast automated protein NMR data collection and assignment by ADAPT-NMR on Bruker spectrometers

    Lee, Woonghee; Hu, Kaifeng; Tonelli, Marco; Bahrami, Arash; Neuhardt, Elizabeth; Glass, Karen C.; Markley, John L.

    2013-11-01

    ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) supports automated NMR data collection and backbone and side chain assignment for [U-13C, U-15N]-labeled proteins. Given the sequence of the protein and data for the orthogonal 2D 1H-15N and 1H-13C planes, the algorithm automatically directs the collection of tilted plane data from a variety of triple-resonance experiments so as to follow an efficient pathway toward the probabilistic assignment of 1H, 13C, and 15N signals to specific atoms in the covalent structure of the protein. Data collection and assignment calculations continue until the addition of new data no longer improves the assignment score. ADAPT-NMR was first implemented on Varian (Agilent) spectrometers [A. Bahrami, M. Tonelli, S.C. Sahu, K.K. Singarapu, H.R. Eghbalnia, J.L. Markley, PLoS One 7 (2012) e33173]. Because of broader interest in the approach, we present here a version of ADAPT-NMR for Bruker spectrometers. We have developed two AU console programs (ADAPT_ORTHO_run and ADAPT_NMR_run) that run under TOPSPIN Versions 3.0 and higher. To illustrate the performance of the algorithm on a Bruker spectrometer, we tested one protein, chlorella ubiquitin (76 amino acid residues), that had been used with the Varian version: the Bruker and Varian versions achieved the same level of assignment completeness (98% in 20 h). As a more rigorous evaluation of the Bruker version, we tested a larger protein, BRPF1 bromodomain (114 amino acid residues), which yielded an automated assignment completeness of 86% in 55 h. Both experiments were carried out on a 500 MHz Bruker AVANCE III spectrometer equipped with a z-gradient 5 mm TCI probe. ADAPT-NMR is available at http://pine.nmrfam.wisc.edu/ADAPT-NMR in the form of pulse programs, the two AU programs, and instructions for installation and use.

  15. 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.

  16. Constraining the S factor of 15N(p,g)16O at Astrophysical Energies

    LeBlanc, P J; Goerres, J; Junker, M; Azuma, R; Beard, M; Bemmerer, D; Best, A; Broggini, C; Caciolli, A; Corvisiero, P; Costantini, H; Couder, M; deBoer, R; Elekes, Z; Falahat, S; Formicola, A; Fulop, Zs; Gervino, G; Guglielmetti, A; Gustavino, C; Gyurky, Gy; Kaeppeler, F; Kontos, A; Kuntz, R; Leiste, H; Lemut, A; Li, Q; Limata, B; Marta, M; Mazzocchi, C; Menegazzo, R; O'Brien, S; Palumbo, A; Prati, P; Roca, V; Rolfs, C; Alvarez, C Rossi; Somorjai, E; Stech, E; Straniero, O; Strieder, F; Tan, W; Terrasi, F; Trautvetter, H P; Uberseder, E; Wiescher, M

    2010-01-01

    The 15N(p,g)16O reaction represents a break out reaction linking the first and second cycle of the CNO cycles redistributing the carbon and nitrogen abundances into the oxygen range. The reaction is dominated by two broad resonances at Ep = 338 keV and 1028 keV and a Direct Capture contribution to the ground state of 16O. Interference effects between these contributions in both the low energy region (Ep < 338 keV) and in between the two resonances (338 15N(p,g)16O reaction has been remeasured covering the energy range from Ep=1800 keV down to 130 keV. The results have been analyzed in the framework of a multi-level R-matrix theory and a S(0) value of 39.6 keV b has been found.

  17. 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

  18. 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

  19. 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.

  20. 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)

  1. 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...

  2. Out-and-back {sup 13}C-{sup 13}C scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS

    Barbet-Massin, Emeline; Pell, Andrew J. [University of Lyon, CNRS/ENS Lyon/UCB Lyon 1, Centre de RMN a Tres Hauts Champs (France); Jaudzems, Kristaps [Latvian Institute of Organic Synthesis (Latvia); Franks, W. Trent; Retel, Joren S. [Leibniz-Institut fuer Molekulare Pharmakologie (Germany); Kotelovica, Svetlana; Akopjana, Inara; Tars, Kaspars [Biomedical Research and Study Center (Latvia); Emsley, Lyndon [University of Lyon, CNRS/ENS Lyon/UCB Lyon 1, Centre de RMN a Tres Hauts Champs (France); Oschkinat, Hartmut [Leibniz-Institut fuer Molekulare Pharmakologie (Germany); Lesage, Anne; Pintacuda, Guido, E-mail: guido.pintacuda@ens-lyon.fr [University of Lyon, CNRS/ENS Lyon/UCB Lyon 1, Centre de RMN a Tres Hauts Champs (France)

    2013-08-15

    We present here {sup 1}H-detected triple-resonance H/N/C experiments that incorporate CO-CA and CA-CB out-and-back scalar-transfer blocks optimized for robust resonance assignment in biosolids under ultra-fast magic-angle spinning (MAS). The first experiment, (H)(CO)CA(CO)NH, yields {sup 1}H-detected inter-residue correlations, in which we record the chemical shifts of the CA spins in the first indirect dimension while during the scalar-transfer delays the coherences are present only on the longer-lived CO spins. The second experiment, (H)(CA)CB(CA)NH, correlates the side-chain CB chemical shifts with the NH of the same residue. These high sensitivity experiments are demonstrated on both fully-protonated and 100 %-H{sup N} back-protonated perdeuterated microcrystalline samples of Acinetobacter phage 205 (AP205) capsids at 60 kHz MAS.

  3. ADAPT-NMR 3.0: utilization of BEST-type triple-resonance NMR experiments to accelerate the process of data collection and assignment

    ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) is a software package whose Bayesian core uses on-the-fly chemical shift assignments to guide data acquisition by non-uniform sampling from a panel of through-bond NMR experiments. The new version of ADAPT-NMR (ADAPT-NMR v3.0) has the option of utilizing 2D tilted-plane versions of 3D fast spectral acquisition with BEST-type pulse sequences, while also retaining the capability of acquiring and processing data from tilted-plane versions of conventional sensitivity-enhanced experiments. The use of BEST experiments significantly reduces data collection times and leads to enhanced performance by ADAPT-NMR

  4. HYPER: A hierarchical algorithm for automatic determination of protein dihedral-angle constraints and stereospecific CβH2 resonance assignments from NMR data

    A new computer program, HYPER, has been developed for automated analysis of protein dihedral angle values and CβH2 stereospecific assignments from NMR data. HYPER uses a hierarchical grid-search algorithm to determine allowed values of φ, Ψ, and χ1 dihedral angles and CβH2 stereospecific assignments based on a set of NMR-derived distance and/or scalar-coupling constraints. Dihedral-angle constraints are valuable for restricting conformational space and improving convergence in three-dimensional structure calculations. HYPER computes the set of φ, Ψ, and χ1dihedral angles and CβH2 stereospecific assignments that are consistent with up to nine intraresidue and sequential distance bounds, two pairs of relative distance bounds, thirteen homo- and heteronuclear scalar coupling bounds, and two pairs of relative scalar coupling constant bounds. The program is designed to be very flexible, and provides for simple user modification of Karplus equations and standard polypeptide geometries, allowing it to accommodate recent and future improved calibrations of Karplus curves. The C code has been optimized to execute rapidly (0.3-1.5 CPU-sec residue-1 using a 5 deg. grid) on Silicon Graphics R8000, R10000 and Intel Pentium CPUs, making it useful for interactive evaluation of inconsistent experimental constraints. The HYPER program has been tested for internal consistency and reliability using both simulated and real protein NMR data sets

  5. 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.)

  6. Resonances

    an impetus or drive to that account: change, innovation, rupture, or discontinuity. Resonances: Historical Essays on Continuity and Change explores the historiographical question of the modes of interrelation between these motifs in historical narratives. The essays in the collection attempt to...... realize theoretical consciousness through historical narrative ‘in practice’, by discussing selected historical topics from Western cultural history, within the disciplines of history, literature, visual arts, musicology, archaeology, philosophy, and theology. The title Resonances indicates the overall...

  7. 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

  8. Assignment of selected hyperfine proton NMR resonances in the met forms of Glycera dibranchiata monomer hemoglobins and comparisons with sperm whale metmyoglobin

    This work indicates a high degree of purity for our preparations of all three of the primary Glycera dibranchiata monomer hemoglobins and details assignments of the heme methyl and vinyl protons in the hyperfine shift region of the ferric (aquo?) protein forms. The assignments were carried out by reconstituting the apoproteins of each component with selectively deuteriated hemes. The results indicate that even though the individual component preparations consist of essentially a single protein, the proton NMR spectra indicate spectroscopic heterogeneity. Evidence is presented for identification and classification of major and minor protein forms that are present in solutions of each component. Finally, in contrast to previous results, a detailed analysis of the proton hyperfine shift patterns of the major and minor forms of each component, in comparison to the major and minor forms of metmyoglobin, leads to the conclusions that the corresponding forms of the proteins from each species have strikingly similar heme-globin contacts and display nearly identical heme electronic structures and coordination numbers

  9. 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

  10. 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)

  11. First measurement of the {sup 18}O(p,{alpha}){sup 15}N cross section at astrophysical energies

    Cognata, M La; Spitaleri, C; Cherubini, S; Gulino, M; Lamia, L; Pizzone, R G; Puglia, S M R; Rapisarda, G G; Romano, S; Sergi, M L [INFN Laboratori Nazionali del Sud, Catania (Italy); Mukhamedzhanov, A; Tribble, R E; Al-Abdullah, T; Banu, A; Goldberg, V [Cyclotron Institute, Texas A and M University, College Station, TX (United States); Coc, A [CSNSM, CNRS/IN2P3, Universite Paris Sud, Orsay (France); Irgaziev, B [GIK Institute of Engineering Sciences and Technology, Topi, District Swabi, N. W. F. P. (Pakistan); Kiss, G G [ATOMKI, Debrecen (Hungary); Mrazek, J [Nuclear Physics Institute of ASCR, Rez near Prague (Czech Republic); Crucilla, V, E-mail: LaCognata@lns.infn.i

    2010-01-01

    The {sup 18}O(p,{alpha}){sup 15}N reaction rate has been deduced by means of the Trojan horse method. For the first time the contribution of the 20 keV resonance has been directly evaluated, giving a value about 35% larger than the one in the literature. Moreover, the present approach has allowed to improve the accuracy by a factor 8.5, as it is based on the measured strength instead of spectroscopic measurements. The contribution of the 90 keV resonance has been also determined, which turned out to be of negligible importance to astrophysics.

  12. 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

  13. Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.

  14. Stable isotope tracking of endangered sea turtles: validation with satellite telemetry and δ15N analysis of amino acids.

    Jeffrey A Seminoff

    Full Text Available Effective conservation strategies for highly migratory species must incorporate information about long-distance movements and locations of high-use foraging areas. However, the inherent challenges of directly monitoring these factors call for creative research approaches and innovative application of existing tools. Highly migratory marine species, such as marine turtles, regularly travel hundreds or thousands of kilometers between breeding and feeding areas, but identification of migratory routes and habitat use patterns remains elusive. Here we use satellite telemetry in combination with compound-specific isotope analysis of amino acids to confirm that insights from bulk tissue stable isotope analysis can reveal divergent migratory strategies and within-population segregation of foraging groups of critically endangered leatherback sea turtles (Dermochelys coriacea across the Pacific Ocean. Among the 78 turtles studied, we found a distinct dichotomy in δ(15N values of bulk skin, with distinct "low δ(15N" and "high δ(15N" groups. δ(15N analysis of amino acids confirmed that this disparity resulted from isotopic differences at the base of the food chain and not from differences in trophic position between the two groups. Satellite tracking of 13 individuals indicated that their bulk skin δ(15N value was linked to the particular foraging region of each turtle. These findings confirm that prevailing marine isoscapes of foraging areas can be reflected in the isotopic compositions of marine turtle body tissues sampled at nesting beaches. We use a Bayesian mixture model to show that between 82 and 100% of the 78 skin-sampled turtles could be assigned with confidence to either the eastern Pacific or western Pacific, with 33 to 66% of all turtles foraging in the eastern Pacific. Our forensic approach validates the use of stable isotopes to depict leatherback turtle movements over broad spatial ranges and is timely for establishing wise conservation

  15. 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)

  16. 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)

  17. 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

  18. 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)

  19. Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: Sequential resonance assignment and implications for protein dynamics and receptor recognition

    Hua, Qingxin (Harvard Medical School, Boston, MA (United States)); Weiss, M.A. (Harvard Medical School, Boston, MA (United States) Massachusetts General Hospital, Boston, MA (United States))

    1991-06-04

    The solution structure and dynamics of human insulin are ivestigated by 2D {sup 1}H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide (B26-B30) insulin. This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three {alpha}-helices and B-chain {beta}-turn) is similar to that observed in the 2-Zn crustal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structues. (3) residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To their knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening.

  20. Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: Sequential resonance assignment and implications for protein dynamics and receptor recognition

    The solution structure and dynamics of human insulin are ivestigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide (B26-B30) insulin. This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three α-helices and B-chain β-turn) is similar to that observed in the 2-Zn crustal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structues. (3) residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To their knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening

  1. 1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis

    Neumoin, A.; Leonchiks, A.; Petit, P.; Vuillard, L.; Pizza, M.; Soriani, M.; Boelens, R.; Bonvin, A.M.J.J.

    2011-01-01

    GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946

  2. 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)

  3. 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.)

  4. 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)

  5. 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)

  6. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly 13C, 15N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  7. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Wasmer, Christian [Harvard Medical School (United States); Bousset, Luc; Sourigues, Yannick [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Schuetz, Anne [ETH Zurich, Physical Chemistry (Switzerland); Loquet, Antoine [Max Planck Institute for Biophysical Chemistry (Germany); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Melki, Ronald, E-mail: melki@lebs.cnrs-gif.fr [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France)

    2011-11-15

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly {sup 13}C, {sup 15}N labeled protein sample, sequential chemical-shift information for 74% of the N, C{alpha}, C{beta} triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  8. 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.)

  9. 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)

  10. 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

  11. 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)

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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)

  17. 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)

  18. BTEC Integrative Assignments.

    Foot, G. E.

    1992-01-01

    To equip electrical engineering students with common and transferable work skills, a program of integrative assignments was created to develop communication and teamwork skills. Discusses assignment components; the log book, a personal account of each assignment; assessment; conversion of "common skills" to competence statements, and performance…

  19. Raman and I.R. spectra of the NH and ND stretching region in polycristalline imidazole, 15N and D substituted analogs

    I.R. and Raman Spectra of imidazole and eleven 15N and D substituted analogs have been analysed, in the region 3300-1800 cm-1. The broad bands with fine structure observed correspond to two spectral features: a) The overall broad bands are attributed to associated NH(D)...N stretching vibrations. Observed νNH 15N shifts are 10 to 15 cm-1 in I.R. which is more than expected for pure νNH. This is probably due to νNH coupling with external modes. b) The fine structure is interpreted as due to interactions by Fermi resonance of νNH(D) with overtones and combinations of internal modes. Considering the temperature and 15N shifts, transmission windows or Evans holes rather than band maximum are found to correspond to overtone and combination values

  20. 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)

  1. Triterpenóides pentacíclicos de Mentha villosa: identificação estrutural e atribuição dos deslocamentos químicos dos átomos de hidrogênio e carbono Pentacyclic triterpenoids of Mentha villosa: structural identification and ¹H and 13C resonance assignments

    Francisco J. Queiroz Monte; Eliete F. de Oliveira; Raimundo Braz Filho

    2001-01-01

    The structures of seven oleanene and ursene triterpenoids (1-7) isolated from aerial parts of Mentha villosa were identified. In addition, the complete ¹H and 13C resonance assignments of these triterpenoids were accomplished using 1D and 2D NMR spectroscopic experiments.

  2. 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...

  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. 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)

  5. 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

  6. 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)

  7. 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)

  8. 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)

  9. 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)

  10. Spectral editing at ultra-fast magic-angle-spinning in solid-state NMR: facilitating protein sequential signal assignment by HIGHLIGHT approach

    This study demonstrates a novel spectral editing technique for protein solid-state NMR (SSNMR) to simplify the spectrum drastically and to reduce the ambiguity for protein main-chain signal assignments in fast magic-angle-spinning (MAS) conditions at a wide frequency range of 40–80 kHz. The approach termed HIGHLIGHT (Wang et al., in Chem Comm 51:15055–15058, 2015) combines the reverse 13C, 15N-isotope labeling strategy and selective signal quenching using the frequency-selective REDOR pulse sequence under fast MAS. The scheme allows one to selectively observe the signals of “highlighted” labeled amino-acid residues that precede or follow unlabeled residues through selectively quenching 13CO or 15N signals for a pair of consecutively labeled residues by recoupling 13CO–15N dipolar couplings. Our numerical simulation results showed that the scheme yielded only ∼15 % loss of signals for the highlighted residues while quenching as much as ∼90 % of signals for non-highlighted residues. For lysine-reverse-labeled micro-crystalline GB1 protein, the 2D 15N/13Cα correlation and 2D 13Cα/13CO correlation SSNMR spectra by the HIGHLIGHT approach yielded signals only for six residues following and preceding the unlabeled lysine residues, respectively. The experimental dephasing curves agreed reasonably well with the corresponding simulation results for highlighted and quenched residues at spinning speeds of 40 and 60 kHz. The compatibility of the HIGHLIGHT approach with fast MAS allows for sensitivity enhancement by paramagnetic assisted data collection (PACC) and 1H detection. We also discuss how the HIGHLIGHT approach facilitates signal assignments using 13C-detected 3D SSNMR by demonstrating full sequential assignments of lysine-reverse-labeled micro-crystalline GB1 protein (∼300 nmol), for which data collection required only 11 h. The HIGHLIGHT approach offers valuable means of signal assignments especially for larger proteins through reducing the