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Sample records for nmr binding constant

  1. Theoretical calculation of the NMR spin-spin coupling constants and the NMR shifts allow distinguishability between the specific direct and the water-mediated binding of a divalent metal cation to guanine.

    Science.gov (United States)

    Sychrovský, Vladimír; Sponer, Jirí; Hobza, Pavel

    2004-01-21

    The calculated intermolecular and intramolecular indirect NMR spin-spin coupling constants and NMR shifts were used for the discrimination between the inner-shell and the outer-shell binding motif of hydrated divalent cations Mg(2+) or Zn(2+) with a guanine base. The intermolecular coupling constants (1)J(X,O6) and (1)J(X,N7) (X = Mg(2+), Zn(2+)) can be unambiguously assigned to the specific inner-shell binding motif of the hydrated cation either with oxygen O6 or with nitrogen N7 of guanine. The calculated coupling constants (1)J(Mg,O6) and (1)J(Zn,O6) were 6.2 and -17.5 Hz, respectively, for the inner-shell complex of cation directly interacting with oxygen O6 of guanine. For the inner-shell coordination of the cation at nitrogen N7, the calculated coupling constants (1)J(Mg,N7) and (1)J(Zn,N7) were 5.6 and -36.5 Hz, respectively. When the binding of the cation is water-mediated, the coupling constant is zero. To obtain reliable shifts in NMR parameters, hydrated guanine was utilized as the reference state. The calculated change of NMR spin-spin coupling constants due to the hydration and coordination of the cation with guanine is caused mainly by the variation of Fermi-contact coupling contribution while the variation of diamagnetic spin-orbit, paramagnetic spin-orbit, and spin-dipolar coupling contributions is small. The change of s-character of guanine sigma bonding, sigma antibonding, and lone pair orbitals upon the hydration and cation coordination (calculated using the Natural Bond Orbital analysis) correlates with the variation of the Fermi-contact term. The calculated NMR shifts delta(N7) of -15.3 and -12.2 ppm upon the coordination of Mg(2+) and Zn(2+) ion are similar to the NMR shift of 19.6 ppm toward the high field measured by Tanaka for N7 of guanine upon the coordination of the Cd(2+) cation (Tanaka, Y.; Kojima, C.; Morita, E. H.; Kasai. Y.; Yamasaki, K.; Ono, A.; Kainosho, M.; Taira, K. J. Am. Chem. Soc. 2002, 124, 4595-4601). The present data

  2. Determination by NMR of the binding constant for the molecular complex between alprostadil and alpha-cyclodextrin. Implications for a freeze-dried formulation.

    Science.gov (United States)

    Ramstad, Tore; Hadden, Chad E; Martin, Gary E; Speaker, Stanley M; Teagarden, Dirk L; Thamann, Thomas J

    2005-05-30

    A binding constant was determined for the complexation reaction between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD). This constant was used to calculate the fraction PGE1 free upon reconstitution of Caverject dual chamber syringe, indicated for the treatment of erectile dysfunction. The determination was based on the measurement of the chemical shift of the C20 methyl protons of PGE1. The observed chemical shift varies as a linear function of the amount of PGE1 bound. The binding constant was obtained from the binding isotherm, a curve of the observed chemical shift versus free ligand (alpha-CD) concentration, through the application of non-linear regression analysis. A value K11 = 966 M(-1) +/- 130 M(-1) (2s), measured at 27 degrees C, was obtained. This value is in good agreement with those reported in the literature. The percent PGE1 free was subsequently calculated for the reconstituted solution and in the corpora cavernosum after injection. The latter showed PGE1 to be delivered essentially quantitatively to the targeted site.

  3. Ab initio calculation of the NMR shielding constants for histamine

    Science.gov (United States)

    Mazurek, A. P.; Dobrowolski, J. Cz.; Sadlej, J.

    1997-12-01

    The gage-independent atomic orbital (GIAO) approach is used within the coupled Hartree-Fock approximation to compute the 1H, 13C and 15N NMR shielding constants in two tautomeric forms of both the histamine molecule and its protonated form. An analysis of the results shows that the protonation on the end of the chain changes its nitrogen shielding constants of the pyridine and pyrrole type. These changes are much higher for the N(3)-H than for the N(1)-H tautomer.

  4. Spin-rotation and NMR shielding constants in HCl

    Energy Technology Data Exchange (ETDEWEB)

    Jaszuński, Michał, E-mail: michal.jaszunski@icho.edu.pl [Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warszawa, Kasprzaka 44 (Poland); Repisky, Michal; Demissie, Taye B.; Komorovsky, Stanislav; Malkin, Elena; Ruud, Kenneth [Centre for Theoretical and Computational Chemistry, University of Tromsø—The Arctic University of Norway, N-9037 Tromsø (Norway); Garbacz, Piotr; Jackowski, Karol; Makulski, Włodzimierz [Laboratory of NMR Spectroscopy, Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw (Poland)

    2013-12-21

    The spin-rotation and nuclear magnetic shielding constants are analysed for both nuclei in the HCl molecule. Nonrelativistic ab initio calculations at the CCSD(T) level of approximation show that it is essential to include relativistic effects to obtain spin-rotation constants consistent with accurate experimental data. Our best estimates for the spin-rotation constants of {sup 1}H{sup 35}Cl are C{sub Cl}  = −53.914 kHz and C{sub H}  = 42.672 kHz (for the lowest rovibrational level). For the chlorine shielding constant, the ab initio value computed including the relativistic corrections, σ(Cl) = 976.202 ppm, provides a new absolute shielding scale; for hydrogen we find σ(H) = 31.403 ppm (both at 300 K). Combining the theoretical results with our new gas-phase NMR experimental data allows us to improve the accuracy of the magnetic dipole moments of both chlorine isotopes. For the hydrogen shielding constant, including relativistic effects yields better agreement between experimental and computed values.

  5. Cation Hydration Constants by Proton NMR: A Physical Chemistry Experiment.

    Science.gov (United States)

    Smith, Robert L.; And Others

    1988-01-01

    Studies the polarization effect on water by cations and anions. Describes an experiment to illustrate the polarization effect of sodium, lithium, calcium, and strontium ions on the water molecule in the hydration spheres of the ions. Analysis is performed by proton NMR. (MVL)

  6. Fast proton exchange in histidine: measurement of rate constants through indirect detection by NMR spectroscopy.

    Science.gov (United States)

    Sehgal, Akansha Ashvani; Duma, Luminita; Bodenhausen, Geoffrey; Pelupessy, Philippe

    2014-05-19

    Owing to its imidazole side chain, histidine participates in various processes such as enzyme catalysis, pH regulation, metal binding, and phosphorylation. The determination of exchange rates of labile protons for such a system is important for understanding its functions. However, these rates are too fast to be measured directly in an aqueous solution by using NMR spectroscopy. We have obtained the exchange rates of the NH3(+) amino protons and the labile NH(ε2) and NH(δ1) protons of the imidazole ring by indirect detection through nitrogen-15 as a function of temperature (272 KExchange rates up to 8.5×10(4) s(-1) could be determined (i.e., lifetimes as short as 12 μs). The three chemical shifts δH(i) of the invisible exchanging protons H(i) and the three one-bond scalar coupling constants (1)J(N,H(i)) could also be determined accurately.

  7. NMR shielding and spin-rotation constants in XCO (X = Ni, Pd, Pt) molecules

    Science.gov (United States)

    Demissie, Taye B.; Jaszuński, Michał; Malkin, Elena; Komorovský, Stanislav; Ruud, Kenneth

    2015-07-01

    Ab initio nonrelativistic and four-component relativistic DFT (density functional theory) methods are combined to study the spin-rotation and absolute nuclear magnetic resonance (NMR) shielding constants of group 10 transition metal monocarbonyls. Good agreement is obtained between the calculated and available experimental data for the spin-rotation constants and shielding spans for PdCO and PtCO. These data allow us to determine accurate absolute chemical shielding constants for all the nuclei, as well as for the unknown spin-rotation constants. We compare the four-component shielding constants with those obtained from the spin-orbit zeroth-order regular approximation, together with an assessment of the performance of different basis sets. For the first time, relativistically optimised basis sets for the heavy atoms used in the four-component calculations are shown to give converged results for both magnetic properties studied. We dedicate this article to the memory of Professor Nicholas C. Handy.

  8. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    . The experimental data were analysed by a computerised curve fitting procedure using equilibrium equations for multiple binding of ligands, containing relative binding constants, valid whether the ligands are truly insoluble or are slightly soluble and irrespective of aggregation in aqueous solution. A best-fit set...... of relative binding constants was found, and subsequently 30 sets of acceptable constants for each set of data in order to evaluate the variation. The data were first fitted by the relative Scatchard's equation, then by the relative, stoichiometric equation. Scatchard's equation is deduced on the presumption...... that cooperativity is absent while the stoichiometric equation is valid even when cooperativity is present. It was found with palmitate as well as with stearate that the two equations fitted the data equally well, and it was concluded that the observations were compatible with absence of cooperativity. The relative...

  9. Indirect NMR spin-spin coupling constants in diatomic alkali halides.

    Science.gov (United States)

    Jaszuński, Michał; Antušek, Andrej; Demissie, Taye B; Komorovsky, Stanislav; Repisky, Michal; Ruud, Kenneth

    2016-12-28

    We report the Nuclear Magnetic Resonance (NMR) spin-spin coupling constants for diatomic alkali halides MX, where M = Li, Na, K, Rb, or Cs and X = F, Cl, Br, or I. The coupling constants are determined by supplementing the non-relativistic coupled-cluster singles-and-doubles (CCSD) values with relativistic corrections evaluated at the four-component density-functional theory (DFT) level. These corrections are calculated as the differences between relativistic and non-relativistic values determined using the PBE0 functional with 50% exact-exchange admixture. The total coupling constants obtained in this approach are in much better agreement with experiment than the standard relativistic DFT values with 25% exact-exchange, and are also noticeably better than the relativistic PBE0 results obtained with 50% exact-exchange. Further improvement is achieved by adding rovibrational corrections, estimated using literature data.

  10. Using NMR Spectroscopy to Investigate the Solution Behavior of Nerve Agents and Their Binding to Acetylcholinesterase

    Science.gov (United States)

    2016-01-01

    USING NMR SPECTROSCOPY TO INVESTIGATE THE SOLUTION BEHAVIOR OF NERVE AGENTS AND THEIR BINDING TO...XX-01-2016 2. REPORT TYPE Final 3. DATES COVERED (From - To) Jan – Jun 2015 4. TITLE AND SUBTITLE Using NMR Spectroscopy to Investigate the...MOLECULAR MOTIONS AND NMR SPECTROSCOPY ...................................................................................................3 4. THE

  11. Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Boonsri, Pornthip [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Herdendorf, Timothy J.; Miziorko, Henry M. [Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110 (United States); Hannongbua, Supa [Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Sem, Daniel S., E-mail: daniel.sem@cuw.edu [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

  12. Microsolvation of methylmercury: structures, energies, bonding and NMR constants ((199)Hg, (13)C and (17)O).

    Science.gov (United States)

    Flórez, Edison; Maldonado, Alejandro F; Aucar, Gustavo A; David, Jorge; Restrepo, Albeiro

    2016-01-21

    Hartree-Fock (HF) and second order perturbation theory (MP2) calculations within the scalar and full relativistic frames were carried out in order to determine the equilibrium geometries and interaction energies between cationic methylmercury (CH3Hg(+)) and up to three water molecules. A total of nine structures were obtained. Bonding properties were analyzed using the Quantum Theory of Atoms In Molecules (QTAIM). The analyses of the topology of electron densities reveal that all structures exhibit a partially covalent HgO interaction between methylmercury and one water molecule. Consideration of additional water molecules suggests that they solvate the (CH3HgOH2)(+) unit. Nuclear magnetic shielding constants σ((199)Hg), σ((13)C) and σ((17)O), as well as indirect spin-spin coupling constants J((199)Hg-(13)C), J((199)Hg-(17)O) and J((13)C-(17)O), were calculated for each one of the geometries. Thermodynamic stability and the values of NMR constants correlate with the ability of the system to directly coordinate oxygen atoms of water molecules to the mercury atom in methylmercury and with the formation of hydrogen bonds among solvating water molecules. Relativistic effects account for 11% on σ((13)C) and 14% on σ((17)O), which is due to the presence of Hg (heavy atom on light atom, HALA effect), while the relativistic effects on σ((199)Hg) are close to 50% (heavy atom on heavy atom itself, HAHA effect). J-coupling constants are highly influenced by relativity when mercury is involved as in J((199)Hg-(13)C) and J((199)Hg-(17)O). On the other hand, our results show that the values of NMR constants for carbon and oxygen, atoms which are connected through mercury (C-HgO), are highly correlated and are greatly influenced by the presence of water molecules. Water molecules introduce additional electronic effects to the relativistic effects due to the mercury atom.

  13. Relativistic force field: parametric computations of proton-proton coupling constants in (1)H NMR spectra.

    Science.gov (United States)

    Kutateladze, Andrei G; Mukhina, Olga A

    2014-09-05

    Spin-spin coupling constants in (1)H NMR carry a wealth of structural information and offer a powerful tool for deciphering molecular structures. However, accurate ab initio or DFT calculations of spin-spin coupling constants have been very challenging and expensive. Scaling of (easy) Fermi contacts, fc, especially in the context of recent findings by Bally and Rablen (Bally, T.; Rablen, P. R. J. Org. Chem. 2011, 76, 4818), offers a framework for achieving practical evaluation of spin-spin coupling constants. We report a faster and more precise parametrization approach utilizing a new basis set for hydrogen atoms optimized in conjunction with (i) inexpensive B3LYP/6-31G(d) molecular geometries, (ii) inexpensive 4-31G basis set for carbon atoms in fc calculations, and (iii) individual parametrization for different atom types/hybridizations, not unlike a force field in molecular mechanics, but designed for the fc's. With the training set of 608 experimental constants we achieved rmsd <0.19 Hz. The methodology performs very well as we illustrate with a set of complex organic natural products, including strychnine (rmsd 0.19 Hz), morphine (rmsd 0.24 Hz), etc. This precision is achieved with much shorter computational times: accurate spin-spin coupling constants for the two conformers of strychnine were computed in parallel on two 16-core nodes of a Linux cluster within 10 min.

  14. Measuring JHH values with a selective constant-time 2D NMR protocol

    Science.gov (United States)

    Lin, Liangjie; Wei, Zhiliang; Lin, Yanqin; Chen, Zhong

    2016-11-01

    Proton-proton scalar couplings play important roles in molecule structure elucidation. However, measurements of JHH values in complex coupled spin systems remain challenging. In this study, we develop a selective constant-time (SECT) 2D NMR protocol with which scalar coupling networks involving chosen protons can be revealed, and corresponding JHH values can be measured through doublets along the F1 dimension. All JHH values within a network of n fully coupled protons can be separately determined with (n - 1) SECT experiments. Additionally, the proposed pulse sequence possesses satisfactory sensitivity and handy implementation. Therefore, it will interest scientists who intend to address structural analyzes of molecules with overcrowded spectra, and may greatly facilitate the applications of scalar-coupling constants in molecule structure studies.

  15. Auto-FACE: an NMR based binding site mapping program for fast chemical exchange protein-ligand systems.

    Directory of Open Access Journals (Sweden)

    Janarthanan Krishnamoorthy

    Full Text Available BACKGROUND: Nuclear Magnetic Resonance (NMR spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQCexperiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. METHODOLOGY/PRINCIPAL FINDINGS: We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues.Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBcl(XL and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3.0 x 10(4 calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12.8 x 10(4. Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker

  16. Investigation of the binding of roxatidine acetate hydrochloride with cyclomaltoheptaose (β-cyclodextrin) using IR and NMR spectroscopy.

    Science.gov (United States)

    Maheshwari, Arti; Sharma, Manisha; Sharma, Deepak

    2011-09-27

    NMR chemical shift changes of the cyclomaltoheptaose (β-cyclodextrin, β-CD) cavity protons as well as roxatidine acetate hydrochloride aromatic ring protons revealed the formation of a RAH-β-CD inclusion complex. Detailed FTIR and NMR spectroscopic ((1)H NMR, COSY, NOESY, ROESY) studies have been done. The stoichiometry of the complex was determined to be 1:1, and the overall binding constant was also determined by Scott's method. The NOESY spectrum confirmed the selective penetration of the aromatic ring of RAH into the β-CD cavity in comparison to that of the piperidine ring. The mode of penetration of the guest into the CD cavity and structure of the complex has been established.

  17. Kinetics of membrane binding and dissociation of 5-fluorouracil by pulsed-field-gradient 19F NMR

    Science.gov (United States)

    Yoshii, Noriyuki; Okamura, Emiko

    2009-06-01

    The kinetics of membrane binding and dissociation of an anticancer drug, 5-fluorouracil (5FU) is quantified by high resolution NMR with the pulsed-field-gradient technique. The 19F NMR signal of 5FU is analyzed at 293-313 K by the solution of Bloch equation with exchange terms. The rate constants of 5FU binding and dissociation are 0.2 and 4.1 s -1 at 303 K. The 5FU motion in the vertical direction to the membrane surface is restricted as compared with the lateral diffusion, judging from the activation energy (57 kJ/mol) larger than the lateral diffusion in membrane (26 kJ/mol [E. Okamura, N. Yoshii, J. Chem. Phys. 129 (2008) 215102]).

  18. Binding of phenol and differently halogenated phenols to dissolved humic matter as measured by NMR spectroscopy.

    Science.gov (United States)

    Smejkalová, Daniela; Spaccini, Riccardo; Fontaine, Barbara; Piccolo, Alessandro

    2009-07-15

    1H- and 19F-NMR measurements of spin-lattice (T1) and spin-spin (T2) relaxationtimes and diffusion ordered spectroscopy (DOSY) were applied to investigate the association of nonsubstituted (phenol (P)) and halogen-substituted (2,4-dichlorophenol (DCP); 2,4,6-trichlorophenol (TCP), and 2,4,6-trifluorophenol (TFP) phenols with a dissolved humic acid (HA). T1 and T2 values for both 1H and 19F in phenols decreased with enhancing HA concentration, indicating reduction in molecular mobility due to formation of noncovalent interactions. Moreover, correlation times (tau c) for different hydrogen and fluorine atoms in phenols showed that anisotropic mobility turned into isotropic motion with HA additions. Changes in relaxation times suggested that DCP and TCP were more extensively bound to HA than P and TFP. This was confirmed by diffusion measurements which showed full association of DCP and TCP to a less amount of HA than that required for entire complexation of P and TFP. Calculated values of binding constants (Ka) reflected the overall NMR behavior, being significantly larger for DCP- and TCP-HA (10.04 +/- 1.32 and 4.47 +/- 0.35 M(-1), respectively) than for P- and TFP-HA complexes (0.57 +/- 0.03 and 0.28 +/- 0.01 M(-1), respectively). Binding increased with decreasing solution pH, thus indicating a dependence on the fraction of protonated form (alpha) of phenols in solution. However, it was found that the hydrophobicity conferred to phenols by chlorine atoms on aromatic rings is a stronger drive than alpha for the phenols repartition within the HA hydrophobic domains.

  19. Importance of triples contributions to NMR spin-spin coupling constants computed at the CC3 and CCSDT levels

    DEFF Research Database (Denmark)

    Faber, Rasmus; Sauer, Stephan P. A.; Gauss, Jürgen

    2017-01-01

    We present the first analytical implementation of CC3 second derivatives using the spin-unrestricted approach. This allows, for the first time, the calculation of nuclear spin-spin coupling constants (SSCC) relevant to NMR spectroscopy at the CC3 level of theory in a fully analytical manner. CC3 ...

  20. Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

    Energy Technology Data Exchange (ETDEWEB)

    Lindfors, Hanna E. [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands); Koning, Peter E. de; Wouter Drijfhout, Jan [Leiden University Medical Centre, Department of Immunohematology and Blood Transfusion (Netherlands); Venezia, Brigida; Ubbink, Marcellus [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands)], E-mail: m.ubbink@chem.leidenuniv.nl

    2008-07-15

    Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

  1. Automated Fragmentation Polarizable Embedding Density Functional Theory (PE-DFT) Calculations of Nuclear Magnetic Resonance (NMR) Shielding Constants of Proteins with Application to Chemical Shift Predictions

    DEFF Research Database (Denmark)

    Svendsen, Casper Steinmann; Bratholm, L.A.; Olsen, Jógvan Magnus Haugaard

    2017-01-01

    Full-protein nuclear magnetic resonance (NMR) shielding constants based on ab initio calculations are desirable, because they can assist in elucidating protein structures from NMR experiments. In this work, we present NMR shielding constants computed using a new automated fragmentation (J. Phys. ...... can obtain a representative subset of snapshots that gives the smallest predicted error, compared to experiment. Finally, we use this subset of snapshots to calculate the NMR shielding constants at the PE-KT3/pcSseg-2 level of theory for all atoms in the protein GB3....

  2. Binding constants of phenylalanine for the four mononucleotides

    Science.gov (United States)

    Khaled, M. A.; Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1984-01-01

    Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: to which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

  3. Tight-binding theory of NMR shifts in topological insulators

    Science.gov (United States)

    Garate, Ion; Boutin, Samuel; Ramirez Ruiz, Jorge

    To date, most experiments in topological insulators have focused on probing the surface states of these materials and suppressing the often inevitable contribution from bulk states. However, the latter are of interest on their own and contain useful information that can be extracted with a local probe like nuclear magnetic resonance (NMR). Recently, 77Se NMR experiments on Bi2Se3 single crystals have reported unusual field-independent linewidths and short spin-echo decays. It is likely that an unexpectedly strong indirect internuclear coupling, characteristic of some inverted band structures, is the cause of these peculiar results. Motivated by this hypothesis, we report on a microscopic theory of NMR shifts and linewidths in Bi2Se3 and Bi2Te3. Our theory provides quantitative estimates for the Knight shift, the orbital shift, the Ruderman-Kittel-Kasuya-Yoshida coupling and the Bloembergen-Rowland coupling. We will compare our findings with the available experimental data Funded by the National Science and Engineering Research Council of Canada, Fonds de Recherche Québécois Nature et Technologies, and Mitacs-Globalink.

  4. Mn(II) binding to human serum albumin: a ¹H-NMR relaxometric study.

    Science.gov (United States)

    Fanali, Gabriella; Cao, Yu; Ascenzi, Paolo; Fasano, Mauro

    2012-12-01

    Human serum albumin (HSA) displays several metal binding sites, participating to essential and toxic metal ions disposal and transport. The major Zn(II) binding site, called Site A, is located at the I/II domain interface, with residues His67, Asn99, His247, and Asp249 contributing with five donor atoms to the metal ion coordination. Additionally, one water molecule takes part of the octahedral coordination geometry. The occurrence of the metal-coordinated water molecule allows the investigation of the metal complex geometry by water (1)H-NMR relaxation, provided that the diamagnetic Zn(II) is replaced by the paramagnetic Mn(II). Here, the (1)H-NMR relaxometric study of Mn(II) binding to HSA is reported. Mn(II) binding to HSA is modulated by Zn(II), pH, and myristate through competitive inhibition and allosteric mechanisms. The body of results indicates that the primary binding site of Zn(II) corresponds to the secondary binding site of Mn(II), i.e. the multimetal binding site A. Excess Zn(II) completely displaces Mn(II) from its primary site suggesting that the primary Mn(II) site corresponds to the secondary Zn(II) site. This uncharacterized site is functionally-linked to FA1; moreover, metal ion binding is modulated by myristate and pH. Noteworthy, water (1)H-NMR relaxometry allowed a detailed analysis of thermodynamic properties of HSA-metal ion complexes.

  5. Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

    Science.gov (United States)

    Armitage, Ian M; Drakenberg, Torbjörn; Reilly, Brian

    2013-01-01

    Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd

  6. Cycloalkane and alicyclic heterocycle complexation by new switchable resorcin[4]arene-based container molecules: NMR and ITC binding studies.

    Science.gov (United States)

    Hornung, Jens; Fankhauser, Daniel; Shirtcliff, Laura D; Praetorius, Antonia; Schweizer, W Bernd; Diederich, François

    2011-10-24

    The synthesis and structural characterization of novel, "molecular basket"-type bridged cavitands is reported. The resorcin[4]arene-based container molecules feature well-defined cavities that bind a wide variety of cycloalkanes and alicyclic heterocycles. Association constants (K(a)) of the 1:1 inclusion complexes were determined by both (1)H NMR and isothermal titration calorimetry (ITC). The obtained K(a) values in mesitylene ranged from 1.7×10(2) M(-1) for cycloheptane up to 1.7×10(7) M(-1) for morpholine. Host-guest complexation by the molecular baskets is generally driven by dispersion interactions, C-H···π interactions of the guests with the aromatic walls of the cavity, and optimal cavity filling. Correlations between NMR-based structural data and binding affinities support that the complexed heterocyclic guests undergo additional polar C-O···C=O, N-H···π, and S···π interactions. The first crystal structure of a cavitand-based molecular basket is reported, providing precise information on the geometry and volume of the inner cavity in the solid state. Molecular dynamic (MD) simulations provided information on the size and conformational preorganization of the cavity in the presence of encapsulated guests. The strongest binding of heterocyclic guests, engaging in polar interactions with the host, was observed at a cavity filling volume of 63 ± 9%.

  7. Rapid NMR screening of RNA secondary structure and binding

    Energy Technology Data Exchange (ETDEWEB)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de [Johann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)

    2015-09-15

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

  8. REDOR NMR of stable-isotope-labeled protein binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, J. [Washington Univ., St. Louis, MO (United States)

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  9. NMR studies of the GTP/GDP binding domain of translation initiation factor IF2

    NARCIS (Netherlands)

    Tishchenko, Evgeny Vladimirovich

    2005-01-01

    Translation Initiation Factor 2 (IF2) plays an important role in the initiation stage of bacterial protein biosynthesis. This protein binds both fMet-tRNA and 30S ribosomal subunit in the presence of GTP, and it stimulates the formation of the 70S initiation complex. The NMR samples of the 15N-, 15N

  10. Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

    DEFF Research Database (Denmark)

    Meier, Sebastian; Beeren, Sophie

    2014-01-01

    to determine binding constants for all other detectable and resolvable hosts. With the use of high-resolution 1H−13C HSQC experiments, complexes of amphiphiles with more than 10 different maltooligosaccharides can be resolved. Hereby, the binding capabilities of a set of structurally related hosts can...

  11. New method for the determination of diffusion constants from partially narrowed NMR lines

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, D.

    1980-01-01

    The effect of atomic and molecular motions on the NMR free-induction decay (FID) and lineshape is investigated theoretically in the intermediate temperature range in which the NMR line is only partially narrowed. It is shown that the FID may be decomposed into the weighted sum of a rigid-lattice (background) contribution and an exponentially decaying part containing all the information on the diffusive or reorientational motions in the crystal in terms of the spin-spin relaxation time T/sub 2/.

  12. Evaluation of three neutral capillary coatings for the determination of analyte-cyclodextrin binding constants by affinity capillary electrophoresis. Application to N,N'-disubstituted piperazine derivatives.

    Science.gov (United States)

    Danel, Cécile; Melnyk, Patricia; Azaroual, Nathalie; Larchanché, Paul-Emmanuel; Goossens, Jean-François; Vaccher, Claude

    2016-07-15

    The performances of three neutral static coatings (hydroxypropyl cellulose, polyethylene oxide and poly(N,N-dimethylacrylamide) have been evaluated in order to determine the binding constants of the complexes formed between four polycationic compounds (piperazine derivatives) and four cyclodextrins of pharmaceutical interest (β-CD, HP-β-CD, Me-β-CD and sulfobutyl ether-β-CD) by affinity capillary electrophoresis. The physically-adsorbed poly(N,N-dimethylacrylamide) coating proves to be the more efficient to mask the silanol groups of the capillary wall since the lowest electroosmotic flow was measured for this coating. Moreover, it drastically reduces the adsorption of the compounds since it allows a correct repeatability of their migration time, higher efficiencies of the peaks and no baseline shift. Then, it was verified for four complexes that this coating allows a correct determination of the binding constants avoiding the CD adsorption which is responsible of an undervaluation of binding constants. The highest binding constants are obtained using the anionic sulfobutyl ether-β-CD (SBE-β-CD). The structure of the complex formed between the tacrine derivative and the SBE-β-CD was further investigated through 2D ROESY NMR experiments and structure-binding constant relationships. Results suggest that the inclusion in the SBE-β-CD cavity occurs through the aliphatic ring portion of the tacrine moiety. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    Science.gov (United States)

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  14. Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

    Science.gov (United States)

    Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2013-09-17

    Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

  15. NMR Studies of a New Binding Mode of the Amino Acid Esters by Porphyrinatozinc(Ⅱ)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The binding mode of the amino acid ethyl esters(guest) by 5-(2-carboxylphenyl)-10,15,20-triphenylporphyrinatozinc(Ⅱ)(host 1) was studied by means of 1H NMR spectra. The binding mode is the hydrogen-bonding between the amino group of the guest and the carboxyl group of host 1 plus the coordination between the zinc atom of porphyrinatozinc(Ⅱ) and the carbonyl group of the guest. This is a novel binding mode of the metalloporphyrin to amino acid derivatives.

  16. Tautomeric ratio and prototropic equilibrium constants of tenoxicam, a 1H and 13C NMR theoretical and experimental study.

    Science.gov (United States)

    Franco-Pérez, Marco; Moya-Hernández, Rosario; Rojas-Hernández, Alberto; Gutiérrez, Atilano; Gómez-Balderas, Rodolfo

    2011-11-24

    The determination of the micro-equilibrium prototropic constants is often a tough task when the tautomeric ratio favors one of the species or when the chemical exchange is not slow enough to allow the quantitative detection of the tautomeric species. There are just few experimental methods available to reveal the constants of the tautomeric micro-equilibriums; its applicability depends on the nature of the tautomeric system. A combination of experimental and quantum chemistry calculated (1)H and (13)C NMR chemical shifts is presented here to estimate the population of the species participating in the tautomeric equilibriums of the tenoxicam, an important anti-inflammatory drug. A multivariate fitting of a fraction-mol-weighted contribution model, for the NMR chemical shifts of the species in solution, was used to find the populations of the tautomers of tenoxicam. To consider and evaluate the effect of the solvent polarity on the tautomers' populations, experimental determinations were carried out in DMSO-d(6), in an equimolar DMSO-H(2)O mixture of deuterated solvents and in D(2)O. Additionally, by employing HYPNMR, it has been possible to refine the acid-base macroscopic constants of tenoxicam.

  17. Sliding and target location of DNA-binding proteins: an NMR view of the lac repressor system.

    Science.gov (United States)

    Loth, Karine; Gnida, Manuel; Romanuka, Julija; Kaptein, Robert; Boelens, Rolf

    2013-05-01

    In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4×10(-12) cm(2)/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.

  18. Sliding and target location of DNA-binding proteins:an NMR view of the lac repressor system

    Energy Technology Data Exchange (ETDEWEB)

    Loth, Karine [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands); Gnida, Manuel; Romanuka, Julija [Paderborn University, Department of Chemistry (Germany); Kaptein, Robert; Boelens, Rolf, E-mail: r.boelens@uu.nl [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)

    2013-05-15

    In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4 Multiplication-Sign 10{sup -12} cm{sup 2}/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.

  19. Sequential sup 1 H NMR assignments and secondary structure of an IgG-binding domain from protein G

    Energy Technology Data Exchange (ETDEWEB)

    Lian, L.Y.; Yang, J.C.; Derrick, J.P.; Sutcliffe, M.J.; Roberts, G.C.K. (Univ. of Leicester (England)); Murphy, J.P.; Goward, C.R.; Atkinson, T. (PHLS Center for Applied Microbiology and Research, Porton Down, Salisbury (England))

    1991-06-04

    Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6,988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by {sup 1}H NMR. Two-dimenstional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the {sup 1}H NMR spectrum of the peptide. Elements of regular secondary structure have been identified by using nuclear Overhauser enhancement, coupling constant, and amide proton exchange data. The secondary structure consists of a central {alpha}-helix (Ala28-Val44), flanked by two portions of {beta}-sheet (Val5-Val26 and Asp45-Lys62). This is a fundamentally different arrangement of secondary structure from that of protein A, which is made up of three consecutive {alpha}-helics in free solution. The authors conclude that the molecular mechanisms underlying the association of protein A and protein G with IgG are different.

  20. Modeling Nonlinear Adsorption with a Single Chemical Parameter: Predicting Chemical Median Langmuir Binding Constants.

    Science.gov (United States)

    Davis, Craig Warren; Di Toro, Dominic M

    2015-07-07

    Procedures for accurately predicting linear partition coefficients onto various sorbents (e.g., organic carbon, soils, clay) are reliable and well established. However, similar procedures for the prediction of sorption parameters of nonlinear isotherm models are not. The purpose of this paper is to present a procedure for predicting nonlinear isotherm parameters, specifically the median Langmuir binding constants, K̃L, obtained utilizing the single-chemical parameter log-normal Langmuir isotherm developed in the accompanying work. A reduced poly parameter linear free energy relationship (pp-LFER) is able to predict median Langmuir binding constants for graphite, charcoal, and Darco granular activated carbon (GAC) adsorption data. For the larger F400 GAC data set, a single pp-LFER model was insufficient, as a plateau is observed for the median Langmuir binding constants of larger molecular volume sorbates. This volumetric cutoff occurs in proximity to the median pore diameter for F400 GAC. A log-linear relationship exists between the aqueous solubility of these large compounds and their median Langmuir binding constants. Using this relationship for the chemicals above the volumetric cutoff and the pp-LFER below the cutoff, the median Langmuir binding constants can be predicted with a root-mean square error for graphite (n = 13), charcoal (n = 11), Darco GAC (n = 14), and F400 GAC (n = 44) of 0.129, 0.307, 0.407, and 0.424, respectively.

  1. NMR shielding and spin–rotation constants of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules

    Energy Technology Data Exchange (ETDEWEB)

    Demissie, Taye B. [Centre for Theoretical and Computational Chemistry Department of Chemistry, UiT – The Arctic University of Norway, N-9037 Tromsø (Norway)

    2015-12-31

    This presentation demonstrates the relativistic effects on the spin-rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants and shielding spans of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules. The results are obtained from calculations performed using density functional theory (non-relativistic and four-component relativistic) and coupled-cluster calculations. The spin-rotation constants are compared with available experimental values. In most of the molecules studied, relativistic effects make an order of magnitude difference on the NMR absolute shielding constants.

  2. Discovery of New E-Selectin Inhibitors by Virtual Screening, Fluorescence Binding Assays, and STD NMR Experiments.

    Science.gov (United States)

    Barra, Pabla A; Jiménez, Verónica A; Gavin, José A; Daranas, Antonio H; Alderete, Joel B

    2016-05-06

    E-selectin is an endothelial protein that participates in the adhesion of metastatic cancer cells, and is therefore a relevant target for antitumor therapeutic intervention. In this work, virtual screening was used to identify new E-selectin inhibitors from a subset of drug-like molecules retrieved from the ZINC database, including the physiological ligand sLe(x) as reference structure (PDB ID: 1G1T). Four hits were chosen and subjected to molecular dynamics simulations and fluorescence binding assays, which led to the determination of experimental dissociation constants between 333 and 1012 μm. The candidate with the highest affinity was studied by saturation transfer difference (STD) NMR experiments and complete relaxation and conformational exchange matrix analysis of saturation transfer (CORCEMA-ST), aimed at identifying the preferable binding mode with E-selectin. Our results revealed that this new inhibitor binds more strongly than sLe(x) in the E-selectin binding site, in good agreement with simulation predictions. These properties will prove valuable for the future design of drugs that target E-selectin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. NMR and molecular modeling of wine tannins binding to saliva proteins: revisiting astringency from molecular and colloidal prospects.

    Science.gov (United States)

    Cala, Olivier; Pinaud, Noël; Simon, Cécile; Fouquet, Eric; Laguerre, Michel; Dufourc, Erick J; Pianet, Isabelle

    2010-11-01

    In organoleptic science, the association of tannins to saliva proteins leads to the poorly understood phenomenon of astringency. To decipher this interaction at molecular and colloidal levels, the binding of 4 procyanidin dimers (B1-4) and 1 trimer (C2) to a human saliva proline-rich peptide, IB7(14), was studied. Interactions have been characterized by measuring dissociation constants, sizes of complexes, number, and nature of binding sites using NMR (chemical shift variations, diffusion-ordered spectroscopy, and saturation transfer diffusion). The binding sites were identified using molecular mechanics, and the hydrophilic/hydrophobic nature of the interactions was resolved by calculating the molecular lipophilicity potential within the complexes. The following comprehensive scheme can be proposed: 1) below the tannin critical micelle concentration (CMC), interaction is specific, and the procyanidin anchorage always occurs on the same three IB7(14) sites. The tannin 3-dimensional structure plays a key role in the binding force and in the tannin's ability to act as a bidentate ligand: tannins adopting an extended conformation exhibit higher affinity toward protein and initiate the formation of a network. 2) Above the CMC, after the first specific hydrophilic interaction has taken place, a random hydrophobic stacking occurs between tannins and proteins. The whole process is discussed in the general frame of wine tannins eliciting astringency.

  4. Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR.

    Science.gov (United States)

    Vasile, Francesca; Reina, José J; Potenza, Donatella; Heggelund, Julie E; Mackenzie, Alasdair; Krengel, Ute; Bernardi, Anna

    2014-08-01

    Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individual's blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence.

  5. THE USE OF HAMMETT CONSTANTS TO UNDERSTAND THE NON-COVALENT BINDING OF AROMATICS

    Directory of Open Access Journals (Sweden)

    Michael Lewis

    2012-04-01

    Full Text Available Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions.

  6. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Craescu Constantin T

    2011-05-01

    Full Text Available Abstract Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

  7. Prediction and dissection of widely-varying association rate constants of actin-binding proteins.

    Directory of Open Access Journals (Sweden)

    Xiaodong Pang

    Full Text Available Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k(a = k(a0 e(-ΔG(el*/k(BT, where k(a0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k(a variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

  8. NMR studies of DNA oligomers and their interactions with minor groove binding ligands

    Energy Technology Data Exchange (ETDEWEB)

    Fagan, Patricia A. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

  9. SOPPA and CCSD vibrational corrections to NMR indirect spin-spin coupling constants of small hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Faber, Rasmus; Sauer, Stephan P. A. [Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø (Denmark)

    2015-12-31

    We present zero-point vibrational corrections to the indirect nuclear spin-spin coupling constants in ethyne, ethene, cyclopropene and allene. The calculations have been carried out both at the level of the second order polarization propagator approximation (SOPPA) employing a new implementation in the DALTON program, at the density functional theory level with the B3LYP functional employing also the Dalton program and at the level of coupled cluster singles and doubles (CCSD) theory employing the implementation in the CFOUR program. Specialized coupling constant basis sets, aug-cc-pVTZ-J, have been employed in the calculations. We find that on average the SOPPA results for both the equilibrium geometry values and the zero-point vibrational corrections are in better agreement with the CCSD results than the corresponding B3LYP results. Furthermore we observed that the vibrational corrections are in the order of 5 Hz for the one-bond carbon-hydrogen couplings and about 1 Hz or smaller for the other couplings apart from the one-bond carbon-carbon coupling (11 Hz) and the two-bond carbon-hydrogen coupling (4 Hz) in ethyne. However, not for all couplings lead the inclusion of zero-point vibrational corrections to better agreement with experiment.

  10. 14N NMR Spectroscopy Study of Binding Interaction between Sodium Azide and Hydrated Fullerene

    Directory of Open Access Journals (Sweden)

    Tamar Chachibaia

    2017-04-01

    Full Text Available Our study is the first attempt to study the interaction between NaN3 and hydrated fullerenes C60 by means of a non-chemical reaction-based approach. The aim is to study deviations of signals obtained by 14N NMR spectroscopy to detect the binding interaction between sodium azide and hydrated fullerene. We considered 14N NMR spectroscopy as one of the most suitable methods for the characterization of azides to show resonance signals corresponding to the three non-equivalent nitrogen atoms. The results demonstrate that there are changes in the chemical shift positions and line-broadening, which are related to the different molar ratios of NaN3:C60 in the samples.

  11. Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy.

    Science.gov (United States)

    Künze, Georg; Köhling, Sebastian; Vogel, Alexander; Rademann, Jörg; Huster, Daniel

    2016-02-05

    The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.

  12. Ligand binding study of human PEBP1/RKIP: interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Laurette Tavel

    Full Text Available BACKGROUND: Human Phosphatidylethanolamine binding protein 1 (hPEBP1 also known as Raf kinase inhibitory protein (RKIP, affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. METHODS/FINDINGS: In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K(D for different ligands. Native mass spectrometry was used as an alternative method for measuring K(D values. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

  13. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    Science.gov (United States)

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off.

  14. NMR J-Coupling Constants of Tl-Pt Bonded Metal Complexes in Aqueous Solution: Ab Initio Molecular Dynamics and Localized Orbital Analysis.

    Science.gov (United States)

    Ducati, Lucas C; Marchenko, Alex; Autschbach, Jochen

    2016-11-21

    The influence of solvent (water) coordination and dynamics on the electronic structure and nuclear magnetic resonance (NMR) indirect spin-spin coupling (J-coupling) constants in a series of Tl-Pt bonded complexes is investigated using Kohn-Sham (KS) Car-Parrinello molecular dynamics (CPMD) and relativistic hybrid KS NMR calculations with and without coordination to water. Coordination of the Tl center by water molecules has a dramatic impact on (1)J(Tl-Pt) and other J-coupling constants. It is shown that a previous computational study of the same complexes using static optimized structures and nonhybrid functionals was correct about the important role of the solvent but obtained reasonable agreement with experimental NMR data because of a cancellation of substantial errors. For example, the CPMD trajectories show that on average the inner coordination shell of Tl is not saturated, as previously assumed, which leads to poor agreement with experiment when the J-coupling constants are averaged over the CPMD trajectories using NMR calculations with nonhybrid functionals. The combination of CPMD with hybrid KS NMR calculations provides a much more realistic computational model that reproduces the large magnitudes of (1)J(Tl-Pt) and the correct trends for other coupling constants. An analysis of (1)J(Tl-Pt) in terms of localized orbitals shows that the presence of coordinating water molecules increases the capacity for covalent interactions between Tl and Pt. There is pronounced multicenter bonding along the metal-metal axis of the complexes.

  15. NMR spin-spin coupling constants in polymethine dyes as polarity indicators.

    Science.gov (United States)

    Murugan, N Arul; Aidas, Kestutis; Kongsted, Jacob; Rinkevicius, Zilvinas; Ågren, Hans

    2012-09-10

    Herein, we explore the use of spin-spin coupling constants (SSCCs) in merocyanine (MCYNE) dyes as indicators of polarity. For this purpose, we use Car-Parrinello hybrid quantum mechanics/molecular mechanics (QM/MM) to determine the structures of MCYNE in solvents of different polarity, followed by computations of the SSCCs by using QM/MM linear-response theory. The molecular geometry of MCYNE switches between neutral, cyanine-like, and zwitterionic depending on the polarity of the solvent. This structural variation is clearly reflected in the proton SSCCs in the polymethine backbone, which are highly sensitive to the dielectric nature of the environment; this mechanism can be used as a "polarity indicator" for different microenvironments. This result is highlighted by computing the SSCCs of the MCYNE probe in the cavity of the beta-lactoglobulin protein. The computed SSCCs clearly indicate a non-polar hydrophobic dielectric nature of this cavity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Importance of Triples Contributions to NMR Spin-Spin Coupling Constants Computed at the CC3 and CCSDT Levels.

    Science.gov (United States)

    Faber, Rasmus; Sauer, Stephan P A; Gauss, Jürgen

    2017-02-14

    We present the first analytical implementation of CC3 second derivatives using the spin-unrestricted approach. This allows, for the first time, the calculation of nuclear spin-spin coupling constants (SSCC) relevant to NMR spectroscopy at the CC3 level of theory in a fully analytical manner. CC3 results for the SSCCs of a number of small molecules and their fluorine substituted derivatives are compared with the corresponding coupled cluster singles and doubles (CCSD) results obtained using specialized basis sets. For one-bond couplings the change when going from CCSD to CC3 is typically 1-3%, but much higher corrections were found for (1)JCN in FCN, 15.7%, and (1)JOF in OF2, 6.4%. The changes vary significantly in the case of multibond couplings, with differences of up to 10%, and even 13.6% for (3)JFH in fluoroacetylene. Calculations at the coupled cluster singles, doubles, and triples (CCSDT) level indicate that the most important contributions arising from connected triple excitations in the coupled cluster expansion are accounted for at the CC3 level. Thus, we believe that the CC3 method will become the standard approach for the calculation of reference values of nuclear spin-spin coupling constants.

  17. Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Chih-Ming [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan (China); Lee, Yuarn-Jang [Section of Infectious Diseases, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 110, Taiwan (China); Wang, Wei-Ting [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Hsu, Chien-Ting [Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Tsai, Jing-Shin [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Wu, Chien-Ming [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan (China); Ou, Keng-Liang [Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); and others

    2011-01-07

    Research highlights: {yields} PD153035 is a DNA intercalator and intercalation occurs only under very low salt concentration. {yields} The minimum distance between adjacent bound PD153035 {approx} 11 bp. {yields} Binding affinity constant for PD153035 is 1.18({+-}0.09) x 10{sup 4} (1/M). {yields} The change of binding free energy of PD153035-DNA interaction is -5.49 kcal mol{sup -1} at 23 {+-} 0.5 {sup o}C. -- Abstract: Accurately predicting binding affinity constant (K{sub A}) is highly required to determine the binding energetics of the driving forces in drug-DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K{sub A} for PD153035, where K{sub A} is determined from the changes in B-form contour length (L) of PD153035-DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K{sub A} = 1.18({+-}0.09) x 10{sup 4} (1/M) at 23 {+-} 0.5 {sup o}C and the minimum distance between adjacent bound PD153035 {approx} 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

  18. Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.

    Science.gov (United States)

    Hein, Christopher; Löhr, Frank; Schwarz, Daniel; Dötsch, Volker

    2017-05-01

    Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial (15) N-, (13) C(α) -, and (13) C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves (15) N, 1-(13) C, 2-(13) C, fully (15) N/(13) C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide (1) H and (15) N resonances need to be obtained, even in cases where sensitivity is the limiting factor. © 2016 Wiley Periodicals, Inc.

  19. Binding and NMR structural studies on indoloquinoline-oligonucleotide conjugates targeting duplex DNA.

    Science.gov (United States)

    Eick, Andrea; Riechert-Krause, Fanny; Weisz, Klaus

    2012-06-20

    An 11-phenyl-indolo[3,2-b]quinoline (PIQ) was tethered through an aminoalkyl linker to the 5'-end of four pyrimidine oligonucleotides with T/C scrambled sequences at their two 5'-terminal positions. Binding to different double-helical DNA targets formed parallel triple helices with a PIQ-mediated stabilization that strongly depends on pH and the terminal base triad at the 5'-triplex-duplex junction. The most effective stabilization was observed with a TAT triplet at the 5'-junction under low pH conditions, pointing to a protonated ligand with a high triplex binding affinity and unfavorable charge repulsions in the case of a terminal C(+)GC triplet at the junction. The latter preference of the PIQ ligand for TAT over CGC is alleviated yet still preserved at higher pH. Intercalation of PIQ at the 5'-triplex-duplex junction as suggested by the triplex melting experiments was confirmed by homonuclear and heteronuclear NMR structural studies on a specifically isotope-labeled triplex. The NMR analysis revealed two coexisting species that only differ by a 180° rotation of the indoloquinoline within the intercalation pocket. NOE-derived molecular models indicate extensive stacking interactions of the indoloquinoline moiety with the TAT base triplet and CG base pair at the junction and a phenyl substituent that is positioned in the major groove and oriented almost perpendicular to the plane of the indoloquinoline.

  20. Discriminating binding and positioning of amphiphiles to lipid bilayers by {sup 1}H NMR

    Energy Technology Data Exchange (ETDEWEB)

    Evanics, F. [Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, Ont., L5L 1C6 (Canada); Prosser, R.S. [Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, Ont., L5L 1C6 (Canada)]. E-mail: sprosser@utm.utoronto.ca

    2005-04-04

    The binding and positioning in lipid bilayers of three well-known drugs--imipramine, nicotine, and caffeine--have been studied using {sup 1}H NMR. The membrane model system consisted of 'fast-tumbling' lipid bicelles, in which a bilayered lipid domain, composed of the unsaturated lipid, 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (DMLPC) was surrounded by a rim of deuterated detergent-like lipids, consisting of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC-d22). Binding and immersion depth information was obtained by three experiments. (1) {sup 1}H chemical shift perturbations, upon transfer of the amphiphiles from water to a bicelle mixture, were used to estimate regions of the amphiphiles that interact with the membrane. (2) Water contact to resolvable protons was measured through a Nuclear Overhauser Effect (NOE) between water and resolvable drug and lipid resonances. In the case of both lipids and membrane bound drugs, positive NOEs with large cross-relaxation rates were measured for most resonances originating from the membrane hydrophilic region, while negative NOEs were observed predominantly to resonances in the hydrophobic region of the membrane. (3) {sup 1}H NMR measurements of oxygen-induced (paramagnetic) spin-lattice relaxation rates, which are known to increase with membrane immersion depth, were used to corroborate conclusions based on chemical shift perturbations and water-ligand NOEs.

  1. Structural, vibrational, NMR, quantum chemical, DNA binding and protein docking studies of two flexible imine oximes

    Indian Academy of Sciences (India)

    YUNUS KAYA

    2016-09-01

    Two flexible imine oxime molecules, namely, 3-(pyridin-2-ylmethylimino)-butan-2-one oxime (HL¹) and 3-(pyridin-2-ylmethylimino)-pentan-2-one oxime (HL²) have been synthesized and characterized by elemental analysis, IR and NMR techniques. The conformational behavior was investigated using the density functional theory (DFT) with the B3LYP method combined with the 6-311++G(d,p) basis set. As a result of the conformational studies, three stable molecules and the most stable conformer were determined for the both imine oximes. The spectroscopic properties such as vibrational and NMR were calculated for the most stable conformer of the HL¹ and HL². The calculation results were applied to simulate infrared spectra of the title compounds, which show good agreement with observed spectra. In addition, the stable three molecules of the both imine oximes have been used to carry out DNA binding and protein docking studies with DNA and protein structures (downloaded from Protein Data Bank) using Discovery Studio 3.5 to find the most preferred binding mode of the ligands inside the DNA and protein cavity.

  2. Constants for mercury binding by organic matter isolates from the Florida Everglades

    Science.gov (United States)

    Benoit, J.M.; Mason, R.P.; Gilmour, C.C.; Aiken, G.R.

    2001-01-01

    Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand approach to estimate conditional stability constants for Hg complexes with DOM isolates collected from Florida Everglades surface waters. The isolates examined were the hydrophobic fraction of DOM from a eutrophic, sulfidic site (F1-HPoA) and the hydrophilic fraction from an oligotrophic, low-sulfide site (2BS-HPiA). Our experimental determinations utilized overall octanol-water partitioning coefficients (Dow) for 203Hg at 0.01 M chloride and across pH and DOM concentration gradients. Use of this radioisotope allowed rapid determinations of Hg concentrations in both water and octanol phases without problems of matrix interference. Conditional stability constants (1 = 0.06, 23??C) were log K??? = 11.8 for F1-HPoA and log K' = 10.6 for 2BS-HPiA. These are similar to previously published stability constants for Hg binding to low-molecular-weight thiols. Further, F1-HPoA showed a pH-dependent decline in Dow that was consistent with models of Hg complexation with thiol groups as the dominant Hg binding sites in DOM. These experiments demonstrate that the DOM isolates are stronger ligands for Hg than chloride ion or ethylenediamine-tetraacetic acid. Speciation calculations indicate that at the DOM concentrations frequently measured in Everglades, 20 to 40 ??M, significant complexation of Hg by DOM would be expected in aerobic (sulfide-free) surface waters. Copyright ?? 2001 Elsevier Science Ltd.

  3. Determination of the binding constant between alprostadil and alpha-cyclodextrin by capillary electrophoresis: implications for a freeze-dried formulation.

    Science.gov (United States)

    Schipper, Benjamin R; Ramstad, Tore

    2005-07-01

    The binding constant between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD) was determined at three temperatures by capillary electrophoresis. alpha-CD is an excipient material in Caverject Dual Chamber Syringe (DCS), added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, the latter of which is critical to product performance. Measurement was made in a pH 7.2 separation buffer to ensure a negative charge on PGE1. The concentration of PGE1 was fixed while the concentration of alpha-CD was varied over a suitable range. As the amount of PGE1 bound to alpha-CD increases, the weighted average of the resultant mobility decreases, thereby allowing a binding isotherm to be generated from which the stability constant was extracted via nonlinear regression analysis. A value of 708 +/- 64 M(-1) was obtained at 27 degrees C, while at physiological temperature (37 degrees C) the value was 537 +/- 27 M(-1). These results compare favorably to values previously obtained by NMR. Calculation of the percent PGE1 free upon reconstitution and injection show it to be near the desired outcome of 100%. Hence, we were able to conclude that the amount of free drug delivered by Caverject DCS is nominally the same as for Caverject S. Po., an earlier-developed product that contains no alpha-CD.

  4. Surface tension method for determining binding constants for cyclodextrin inclusion complexes of ionic surfactants

    Energy Technology Data Exchange (ETDEWEB)

    Dharmawardana, U.R.; Christian, S.D.; Tucker, E.E.; Taylor, R.W.; Scamehorn, J.F. (Univ. of Oklahoma, Norman, OK (United States))

    1993-09-01

    A new method has been developed for determining binding constants of complexes of cyclodextrins with surface-active compounds, including water-soluble ionic surfactants. The technique requires measuring the change in surface tension caused by addition of a cyclodextrin (CD) to aqueous solutions of the surfactant; the experimental results lead directly to inferred values of the thermodynamic activity of the surfactant. Surface tension results are reported for three different surfactants sodium dodecyl sulfate (SDS), cetylpyridinium chloride (CPC), and cetyltrimethylammonium bromide (CTAB) in the presence and in the absence of added [beta]-CD. Data for CPC have been obtained at surfactant concentrations below and above the critical micelle concentration. Correlations between surface tension and surfactant activity are expressed by the Szyszkowski equation, which subsumes the Langmuir adsorption model and the Gibbs equation. It is observed that the surface tension increases monotonically as [beta]-cyclodextrin is added to ionic surfactant solutions. At concentrations of CD well in excess of the surfactant concentration, the surface tension approaches that of pure water, indicating that neither the surfactant-CD complexes nor CD itself are surface active. Binding constants are inferred from a model that incorporates the parameters of the Szyszkowski equation and mass action constants relating to the formation of micelles from monomers of the surfactant and the counterion. Evidence is given that two molecules of CD can complex the C-16 hydrocarbon chain of the cetyl surfactants. 30 refs., 5 figs., 1 tab.

  5. Polypharmacotherapy in rheumatology: 1H NMR analysis of binding of phenylbutazone and methotrexate to serum albumin

    Science.gov (United States)

    Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.; Bojko, B.; Szkudlarek-Haśnik, A.; Knopik, M.; Sułkowski, W. W.

    2011-05-01

    The influence of phenylbutazone (Phe) and methotrexate (MTX) on binding of MTX and Phe to human (HSA) and bovine (BSA) serum albumin in the low-affinity binding sites is investigated. The strength and kind of interactions between serum albumin (SA) and drugs used in combination therapy were found using 1H NMR spectroscopy. A stoichiometric molar ratios for Phe-SA and MTX-SA complexes are 36:1 and 31:1, respectively. It appeared these molar ratios are higher for the ternary systems than it were in the binary ones. The presence of the additional drug (MTX or Phe) causes the increase of an affinity of albumin towards Phe and MTX. It was found that the aliphatic groups of MTX are more resistant to the influence of Phe on the MTX-SA complex than the aromatic rings. The results showed the important impact of another drug (MTX or Phe) on the affinity of SA towards Phe and MTX in the low-affinity binding sites. This work is a subsequent part of the spectroscopic study on Phe-MTX-SA interactions (Maciążek-Jurczyk, 2009 [1]).

  6. Solution NMR structure and histone binding of the PHD domain of human MLL5.

    Directory of Open Access Journals (Sweden)

    Alexander Lemak

    Full Text Available Mixed Lineage Leukemia 5 (MLL5 is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3, the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide 'reader' domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.

  7. NMR and Mutational Identification of the Collagen-Binding Site of the Chaperone Hsp47

    Science.gov (United States)

    Yagi-Utsumi, Maho; Yoshikawa, Sumi; Yamaguchi, Yoshiki; Nishi, Yohei; Kurimoto, Eiji; Ishida, Yoshihito; Homma, Takayuki; Hoseki, Jun; Nishikawa, Yoshimi; Koide, Takaki; Nagata, Kazuhiro; Kato, Koichi

    2012-01-01

    Heat shock protein 47 (Hsp47) acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective 15N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases. PMID:23049894

  8. NMR and mutational identification of the collagen-binding site of the chaperone Hsp47.

    Directory of Open Access Journals (Sweden)

    Maho Yagi-Utsumi

    Full Text Available Heat shock protein 47 (Hsp47 acts as a client-specific chaperone for collagen and plays a vital role in collagen maturation and the consequent embryonic development. In addition, this protein can be a potential target for the treatment of fibrosis. Despite its physiological and pathological importance, little is currently known about the collagen-binding mode of Hsp47 from a structural aspect. Here, we describe an NMR study that was conducted to identify the collagen-binding site of Hsp47. We used chicken Hsp47, which has higher solubility than its human counterpart, and applied a selective (15N-labeling method targeting its tryptophan and histidine residues. Spectral assignments were made based on site-directed mutagenesis of the individual residues. By inspecting the spectral changes that were observed upon interaction with a trimeric collagen peptide and the mutational data, we successfully mapped the collagen-binding site in the B/C β-barrel domain and a nearby loop in a 3D-homology model based upon a serpin fold. This conclusion was confirmed by mutational analysis. Our findings provide a molecular basis for the design of compounds that target the interaction between Hsp47 and procollagen as therapeutics for fibrotic diseases.

  9. Dynamics, NMR parameters and hyperfine coupling constants of the Fe3O4(1 0 0)-water interface: Implications for MRI probes

    Science.gov (United States)

    Gonçalves, Mateus A.; Peixoto, Fernando C.; da Cunha, Elaine F. F.; Ramalho, Teodorico C.

    2014-08-01

    Magnetite is an iron oxide widely used as contrast agent in MRI, receiving considerable interest from nanoscience and nanotechnology. In this work, the face 1 0 0 of the magnetite structure was studied with water in order to obtain 1H hyperfine coupling constants (HFCCs). Molecular dynamics (MD) calculations were performed using the ReaxFF program and for statistical inefficiency, structures were selected for HFCC and NMR calculations. From our theoretical findings, the magnetite in solution considerably increases the 1H HFCC of water molecules. From our results, it is essential to incorporate the dynamics and solvent effects into NMR calculations of relaxation parameters.

  10. Understanding the interaction between trivalent lanthanide ions and stereoregular polymethacrylates through luminescence, binding isotherms, NMR, and interaction with cetylpyridinium chloride.

    Science.gov (United States)

    Kogej, Ksenija; Fonseca, Sofia M; Rovisco, José; Azenha, M Emília; Ramos, M Luísa; Seixas de Melo, J Sérgio; Burrows, Hugh D

    2013-11-26

    Complexation of isotactic, syndiotactic, and atactic poly(methacrylic acid), PMA, with trivalent lanthanide ions has been studied in water at a degree of neutralization 0.5. Metal ion binding is shown by quenching of cerium(III) fluorescence, enhancement of Tb(III) luminescence, and lanthanide-induced line broadening in the PMA (1)H NMR spectra. Comparison with lanthanide-acetate complexation suggests carboxylate binds in a bidentate fashion, while Ce(III) luminescence quenching suggests an ≈3:1 carboxylate:metal ion stoichiometry, corresponding to charge neutralization. The presence of both free and bound Ce(III) cations in PMA solutions is confirmed from luminescence decays. Studies of Tb(3+) luminescence lifetime in H2O and D2O solutions show complexation is accompanied by loss of 5-6 water molecules, indicating that each bidentate carboxylate replaces two coordinated water molecules. The behavior depends on pH and polyelectrolyte stereoregularity, and stronger binding is observed with isotactic polyelectrolyte. Binding of cetylpyridinium chloride, CPC, in these systems is studied by luminescence, NMR, and potentiometry. NMR and Tb(3+) luminescence lifetime studies show the strongest binding with the isotactic polymer. Binding of surfactant to poly(methacrylate) in the presence of lanthanides is noncooperative, i.e., it binds to the free sites; binding isotherms in the presence of lanthanides are shifted to higher free surfactant concentrations, compared with sodium ions, have lower slopes and show a clear two-step binding mechanism. While CPC readily replaces the Na(+) ions of poly(methacrylate) and binds very strongly (low critical association concentrations), exchange is much more difficult with the strongly bound trivalent lanthanide ions. Effects of tacticity are seen, with surfactant interacting most strongly with isotactic chains in the initial stages of binding, while in the final stages of binding the interaction is strongest with atactic poly(methacrylate).

  11. Studies on the binding of modest modulators of the human enzyme, Sirtuin 6, using STD-NMR.

    Science.gov (United States)

    Bolívar, Beatriz E; Welch, John T

    2017-02-21

    Pyrazinamide (PZA), an essential constituent of short course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 via different mechanisms and binding sites, as suggested by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR). Available experimental evidence, such as that derived from crystal structures and kinetic experiments, is of only limited utility in the elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For example, crystallographic structural analysis of sirtuin binding sites does not illuminate differences in binding affinities among sirtuins or capture details of the dynamic process. STD NMR led to findings that not only agreed with the binding kinetics experiments, but also yielded insights into the binding process. The data also illustrate the geometry of the ligand binding epitopes in solution for both the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates may be possible. These derivatives may be accessible by employing the malleability of this scaffold to assist in the identification by STD-NMR of the motifs that interact with the holo- and apo-enzymes in solution.

  12. Contact and dipolar contributions to lanthanide-induced NMR shifts of amino acid and peptide models for calcium binding sites in proteins

    Science.gov (United States)

    Shelling, Judith G.; Bjornson, Michele E.; Hodges, Robert S.; Taneja, Ashok K.; Sykes, Brian D.

    1H nuclear magnetic resonance has been employed to study the binding of Nα-acetyl- L-aspartic acid and Nα-acetyl- L-aspartyl- L-glycyl- L-aspartylamide to the series of six lanthanide ions Dy 3+ through Lu 3+. Values for the dissociation constants and the maximum lanthanide-induced shifts were obtained by fitting the titration data for each metal ion to appropriate binding curves. The shifts were separated into contact and dipolar terms without prior knowledge of the symmetry of the complex or the orientation of the principle axis system of the magnetic susceptibility tensor. The results indicate the contact shifts in 1H NMR are not always negligible, and that Yb 3+ appears to be the best calcium analog for structural studies when the contact interaction is significant.

  13. Tight-binding theory of NMR shifts in topological insulators Bi2Se3 and Bi2Te3

    Science.gov (United States)

    Boutin, Samuel; Ramírez-Ruiz, Jorge; Garate, Ion

    2016-09-01

    Motivated by recent nuclear magnetic resonance (NMR) experiments, we present a microscopic s p3 tight-binding model calculation of the NMR shifts in bulk Bi2Se3 and Bi2Te3 . We compute the contact, dipolar, orbital and core polarization contributions to the carrier-density-dependent part of the NMR shifts in 209Bi,125Te, and 77Se. The spin-orbit coupling and the layered crystal structure result in a contact Knight shift with strong uniaxial anisotropy. Likewise, because of spin-orbit coupling, dipolar interactions make a significant contribution to the isotropic part of the NMR shift. The contact interaction dominates the isotropic Knight shift in 209Bi NMR, even though the electronic states at the Fermi level have a rather weak s -orbital character. In contrast, the contribution from the contact hyperfine interaction to the NMR shift of 77Se and 125Te is weak compared to the dipolar and orbital shifts therein. In all cases, the orbital shift is at least comparable to the contact and dipolar shifts, while the shift due to core polarization is subdominant (except for Te nuclei located at the inversion centers). By artificially varying the strength of spin-orbit coupling, we evaluate the evolution of the NMR shift across a band inversion but find no clear signature of the topological transition.

  14. Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments.

    Science.gov (United States)

    Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F

    2013-02-12

    Phenylalanine hydroxylase (PheH) catalyzes the key step in the catabolism of dietary phenylalanine, its hydroxylation to tyrosine using tetrahydrobiopterin (BH(4)) and O(2). A complete kinetic mechanism for PheH was determined by global analysis of single-turnover data in the reaction of PheHΔ117, a truncated form of the enzyme lacking the N-terminal regulatory domain. Formation of the productive PheHΔ117-BH(4)-phenylalanine complex begins with the rapid binding of BH(4) (K(d) = 65 μM). Subsequent addition of phenylalanine to the binary complex to form the productive ternary complex (K(d) = 130 μM) is approximately 10-fold slower. Both substrates can also bind to the free enzyme to form inhibitory binary complexes. O(2) rapidly binds to the productive ternary complex; this is followed by formation of an unidentified intermediate, which can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s(-1). Formation of the 4a-hydroxypterin and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines k(cat). Similar reactions using 6-methyltetrahydropterin indicate a preference for the physiological pterin during hydroxylation.

  15. NMR studies of the AMP-binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Kuby, S.A.; Mildvan, A.S.

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr/sup 3 +/AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T/sub 1/ method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine.

  16. NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9.

    Science.gov (United States)

    Hannan, J. P.; Whittaker, S. B.; Davy, S. L.; Kühlmann, U. C.; Pommer, A. J.; Hemmings, A. M.; James, R.; Kleanthous, C.; Moore, G. R.

    1999-01-01

    Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+. PMID:10452617

  17. NMr studies of the AMP binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Kuby, S.A.; Fry, D.C.; Mildvan, A.S.

    1986-05-01

    The authors recently located by NMR the MgATP binding site on adenylate kinase correcting the proposed location for this site based on X-ray studies of the binding of salicylate. To determine the conformation and location of the other substrate, they have determined distances from Cr/sup 3 +/ AMPPCP to 6 protons and to the phosphorus atom of AMP on adenylate kinase using the paramagnetic-probe-T/sub 1/ method. They have also used time-dependent NOEs to measure five interproton distances on AMP, permitting evaluation of the conformation of enzyme-bound AMP and its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high-anti glycosyl torsional angle (X = 110/sup 0/), a 3'-endo sugar pucker (delta = 105/sup 0/), and a gauche-trans orientation about the C/sub 4/'-C/sub 5/' bond (..gamma.. = 180/sup 0/). The distance from Cr/sup 3 +/ to the phosphorus of AMP is 6.4 +/- 0.3 A, indicating a reaction coordinate distance of greater than or equal to A which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP were detected. These constraints, together with the conformation of AMP and the X-ray structure of the enzyme, suggest proximity (less than or equal to A) of AMP to leu 116, arg 171, val 173, gln 185, thr 188, and asp 191.

  18. Ratiometric analysis in hyperpolarized NMR (I): test of the two-site exchange model and the quantification of reaction rate constants.

    Science.gov (United States)

    Li, Lin Z; Kadlececk, Stephen; Xu, He N; Daye, Dania; Pullinger, Benjamin; Profka, Harrilla; Chodosh, Lewis; Rizi, Rahim

    2013-10-01

    Conventional methods for the analysis of in vivo hyperpolarized (13) C NMR data from the lactate dehydrogenase (LDH) reaction usually make assumptions on the stability of rate constants and/or the validity of the two-site exchange model. In this study, we developed a framework to test the validity of the assumption of stable reaction rate constants and the two-site exchange model in vivo via ratiometric fitting of the time courses of the signal ratio L(t)/P(t). Our analysis provided evidence that the LDH enzymatic kinetics observed by hyperpolarized NMR are in near-equilibrium and satisfy the two-site exchange model for only a specific time window. In addition, we quantified both the forward and reverse exchange rate constants of the LDH reaction for the transgenic and mouse xenograft models of breast cancer using the ratio fitting method developed, which includes only two modeling parameters and is less sensitive to the influence of instrument settings/protocols, such as flip angles, degree of polarization and tracer dosage. We further compared the ratio fitting method with a conventional two-site exchange modeling method, i.e. the differential equation fitting method, using both the experimental and simulated hyperpolarized NMR data. The ratio fitting method appeared to fit better than the differential equation fitting method for the reverse rate constant on the mouse tumor data, with less relative errors on average, whereas the differential equation fitting method also resulted in a negative reverse rate constant for one tumor. The simulation results indicated that the accuracy of both methods depends on the width of the transport function, noise level and rate constant ratio; one method may be more accurate than the other based on the experimental/biological conditions aforementioned. We were able to categorize our tumor models into specific conditions of the computer simulation and to estimate the errors of rate quantification. We also discussed possible

  19. Temperature dependence of (+)-catechin pyran ring proton coupling constants as measured by NMR and modeled using GMMX search methodology

    Science.gov (United States)

    Fred L. Tobiason; Stephen S. Kelley; M. Mark Midland; Richard W. Hemingway

    1997-01-01

    The pyran ring proton coupling constants for (+)-catechin have been experimentally determined in deuterated methanol over a temperature range of 213 K to 313 K. The experimental coupling constants were simulated to 0.04 Hz on the average at a 90 percent confidence limit using a LAOCOON method. The temperature dependence of the coupling constants was reproduced from the...

  20. Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

    Science.gov (United States)

    Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming Hofmann; Mortensen, Jonas Sigurd; Geiger, Michel-Andreas; van Rossum, Barth-Jan; Oschkinat, Hartmut; Gether, Ulrik; Teilum, Kaare; Loland, Claus J

    2017-01-01

    The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT. DOI: http://dx.doi.org/10.7554/eLife.19314.001 PMID:28117663

  1. Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR.

    Science.gov (United States)

    Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming Hofmann; Mortensen, Jonas Sigurd; Geiger, Michel-Andreas; van Rossum, Barth-Jan; Oschkinat, Hartmut; Gether, Ulrik; Teilum, Kaare; Loland, Claus J

    2017-01-24

    The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

  2. Vicinal 1H-1H NMR coupling constants from density functional theory as reliable tools for stereochemical analysis of highly flexible multichiral center molecules.

    Science.gov (United States)

    López-Vallejo, Fabian; Fragoso-Serrano, Mabel; Suárez-Ortiz, Gloria Alejandra; Hernández-Rojas, Adriana C; Cerda-García-Rojas, Carlos M; Pereda-Miranda, Rogelio

    2011-08-05

    A protocol for stereochemical analysis, based on the systematic comparison between theoretical and experimental vicinal (1)H-(1)H NMR coupling constants, was developed and applied to a series of flexible compounds (1-8) derived from the 6-heptenyl-5,6-dihydro-2H-pyran-2-one framework. The method included a broad conformational search, followed by geometry optimization at the DFT B3LYP/DGDZVP level, calculation of the vibrational frequencies, thermochemical parameters, magnetic shielding tensors, and the total NMR spin-spin coupling constants. Three scaling factors, depending on the carbon atom hybridizations, were found for the (1)H-C-C-(1)H vicinal coupling constants: f((sp3)-(sp3)) = 0.910, f((sp3)-(sp2)) = 0.929, and f((sp2)-(sp2))= 0.977. A remarkable correlation between the theoretical (J(pre)) and experimental (1)H-(1)H NMR (J(exp)) coupling constants for spicigerolide (1), a cytotoxic natural product, and some of its synthetic stereoisomers (2-4) demonstrated the predictive value of this approach for the stereochemical assignment of highly flexible compounds containing multiple chiral centers. The stereochemistry of two natural 6-heptenyl-5,6-dihydro-2H-pyran-2-ones (14 and 15) containing diverse functional groups in the heptenyl side chain was also analyzed by application of this combined theoretical and experimental approach, confirming its reliability. Additionally, a geometrical analysis for the conformations of 1-8 revealed that weak hydrogen bonds substantially guide the conformational behavior of the tetraacyloxy-6-heptenyl-2H-pyran-2-ones.

  3. Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions.

    Science.gov (United States)

    Joseph, Prem Raj B; Mosier, Philip D; Desai, Umesh R; Rajarathnam, Krishna

    2015-11-15

    Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8-GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer-GAG interactions and function.

  4. Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

    Science.gov (United States)

    Steginsky, C A; Beale, J M; Floss, H G; Mayer, R M

    1992-02-17

    The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.

  5. NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function.

    Science.gov (United States)

    Sharma, Alok K; Birrane, Gabriel; Anklin, Clemens; Rigby, Alan C; Alper, Seth L

    2017-04-28

    Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. (15)N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS·LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Benchmarking density-functional theory calculations of NMR shielding constants and spin-rotation constants using accurate coupled-cluster calculations.

    Science.gov (United States)

    Teale, Andrew M; Lutnæs, Ola B; Helgaker, Trygve; Tozer, David J; Gauss, Jürgen

    2013-01-14

    Accurate sets of benchmark nuclear-magnetic-resonance shielding constants and spin-rotation constants are calculated using coupled-cluster singles-doubles (CCSD) theory and coupled-cluster singles-doubles-perturbative-triples [CCSD(T)] theory, in a variety of basis sets consisting of (rotational) London atomic orbitals. The accuracy of the calculated coupled-cluster constants is established by a careful comparison with experimental data, taking into account zero-point vibrational corrections. Coupled-cluster basis-set convergence is analyzed and extrapolation techniques are employed to estimate basis-set-limit quantities, thereby establishing an accurate benchmark data set. Together with the set provided for rotational g-tensors and magnetizabilities in our previous work [O. B. Lutnæs, A. M. Teale, T. Helgaker, D. J. Tozer, K. Ruud, and J. Gauss, J. Chem. Phys. 131, 144104 (2009)], it provides a substantial source of consistently calculated high-accuracy data on second-order magnetic response properties. The utility of this benchmark data set is demonstrated by examining a wide variety of Kohn-Sham exchange-correlation functionals for the calculation of these properties. None of the existing approximate functionals provide an accuracy competitive with that provided by CCSD or CCSD(T) theory. The need for a careful consideration of vibrational effects is clearly illustrated. Finally, the pure coupled-cluster results are compared with the results of Kohn-Sham calculations constrained to give the same electronic density. Routes to future improvements are discussed in light of this comparison.

  7. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  8. Does aluminium bind to histidine? An NMR investigation of amyloid β12 and amyloid β16 fragments.

    Science.gov (United States)

    Narayan, Priya; Krishnarjuna, Bankala; Vishwanathan, Vinaya; Jagadeesh Kumar, Dasappa; Babu, Sudhir; Ramanathan, Krishna Venkatachala; Easwaran, Kalpathy Ramaier Katchap; Nagendra, Holenarasipur Gundurao; Raghothama, Srinivasarao

    2013-07-01

    Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N-terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C-terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium-binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal-binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets. © 2013 John Wiley & Sons A/S.

  9. NMR studies of recombinant Coprinus peroxidase and three site-directed mutants. Implications for peroxidase substrate binding.

    Science.gov (United States)

    Veitch, N C; Tams, J W; Vind, J; Dalbøge, H; Welinder, K G

    1994-06-15

    Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild-type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one- and two-dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem-linked residues obtained for the cyanide-ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese-dependent peroxidases and extend previously reported NMR data for fungal CIP. The three mutants examined by NMR spectroscopy comprised site-specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side-chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.

  10. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    Full Text Available BACKGROUND: Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. METHODS/FINDINGS: In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. CONCLUSIONS/SIGNIFICANCE: This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  11. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  12. Absorption spectroscopy and binding constants for first-row transition metal complexes of a DOPA-containing peptide.

    Science.gov (United States)

    Sever, Mary J; Wilker, Jonathan J

    2006-02-14

    A diverse array of biological systems incorporate 3,4-dihydroxyphenlyalanine (DOPA) into proteins and small molecules for cross-linking and material generation. Marine worm eggshells, sea squirt wound plugs, and marine mussel adhesives may all be formed by combining DOPA-containing molecules with high levels of metals. In order to provide model systems for characterizing these biomaterials, we carried out a study on metal binding to a DOPA-containing peptide. Ultraviolet-visible absorption spectra are presented for the AdopaTP peptide binding to Fe3+, V3+, VO2+, Mn3+, Ti4+, Cu2+, Co2+, and Ni2+ in mono, bis, and where applicable, tris coordination modes. Association constants were determined for selected metal ions binding to the peptide. In general, the spectroscopic and binding properties of this DOPA-containing peptide were found to be similar to those of catechol.

  13. Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with β-cyclodextrin by affinity capillary electrophoresis.

    Science.gov (United States)

    Šolínová, Veronika; Mikysková, Hana; Kaiser, Martin Maxmilián; Janeba, Zlatko; Holý, Antonín; Kašička, Václav

    2016-01-01

    Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)-enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β-cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)-enantiomers of ANPs-based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0-25 mM) of βCD. The apparent binding constants of the complexes of (R,S)-enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)-enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of (R,S)-enantiomers of ANPs with βCD have been found to be relatively weak - their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3-46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3-55.2 L/mol.

  14. Electrostatic interaction between oxysterol-binding protein and VAMP-associated protein A revealed by NMR and mutagenesis studies.

    Science.gov (United States)

    Furuita, Kyoko; Jee, JunGoo; Fukada, Harumi; Mishima, Masaki; Kojima, Chojiro

    2010-04-23

    Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F), and the complex. Our results show that OSBP(F) is disordered in the free state, and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain.

  15. Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies*

    Science.gov (United States)

    Furuita, Kyoko; Jee, JunGoo; Fukada, Harumi; Mishima, Masaki; Kojima, Chojiro

    2010-01-01

    Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-AMSP) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBPF) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-AMSP, OSBPF, and the complex. Our results show that OSBPF is disordered in the free state, and VAP-AMSP and OSBPF form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain. PMID:20178991

  16. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose.

    Science.gov (United States)

    Thorn, K A; Kennedy, K R

    2002-09-01

    The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.

  17. Determination of the 14N quadrupole coupling constant of nitroxide spin probes by W-band ELDOR-detected NMR

    Science.gov (United States)

    Florent, Marc; Kaminker, Ilia; Nagarajan, Vijayasarathi; Goldfarb, Daniella

    2011-06-01

    Nitroxide spin probe electron paramagnetic resonance (EPR) has proven to be a very successful method to probe local polarity and solvent hydrogen bonding properties at the molecular level. The g xx and the 14N hyperfine A zz principal values are the EPR parameters of the nitroxide spin probe that are sensitive to these properties and are therefore monitored experimentally. Recently, the 14N quadrupole interaction of nitroxides has been shown to be also highly sensitive to polarity and H-bonding (A. Savitsky et al., J. Phys. Chem. B 112 (2008) 9079). High-field electron spin echo envelope modulation (ESEEM) was used successfully to determine the P xx and P yy principal components of the 14N quadrupole tensor. The P zz value was calculated from the traceless character of the quadrupole tensor. We introduce here high-field (W-band, 95 GHz, 3.5 T) electron-electron double resonance (ELDOR)-detected NMR as a method to obtain the 14N P zz value directly, together with A zz. This is complemented by W-band hyperfine sublevel correlation (HYSCORE) measurements carried out along the g xx direction to determine the principal P xx and P yy components. Through measurements of TEMPOL dissolved in solvents of different polarities, we show that A zz increases, while | P zz| decreases with polarity, as predicted by Savitsky et al.

  18. Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy.

    Science.gov (United States)

    Barthelmes, Dominic; Gränz, Markus; Barthelmes, Katja; Allen, Karen N; Imperiali, Barbara; Prisner, Thomas; Schwalbe, Harald

    2015-11-01

    We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1β (IL1β) and measured (1)H, (15)N-pseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Δχ-tensors associated with each Tm(3+)-loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1β-S2R2 and the respective single-loop-LBT constructs IL1β-S2, IL1β-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd(3+) as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

  19. Investigation of the NMR spin-spin coupling constants across the hydrogen bonds in ubiquitin: the nature of the hydrogen bond as reflected by the coupling mechanism.

    Science.gov (United States)

    Tuttle, Tell; Kraka, Elfi; Wu, Anan; Cremer, Dieter

    2004-04-28

    The indirect scalar NMR spin-spin coupling constants across the H-bonds of the protein ubiquitin were calculated, including the Fermi contact, the diamagnetic spin-orbit, the paramagnetic spin-orbit, and the spin dipole term, employing coupled perturbed density functional theory in combination with the B3LYP functional and different basis sets: (9s,5p,1d/5s,1p)[6s,4p,1d/3s,1p] and (11s,7p,2d/5s,1p)[7s,6p,2d/4s,2p]. Four different models based on either the crystal or the aqueous solution structure of ubiquitin were used to describe H-bonding for selected residue pairs of ubiquitin. Calculated and measured 3hJ(NC') coupling constants differ depending on the model used, which is due to the fact that the geometry of ubiquitin is different in the solid state and in aqueous solution. Also, conformational averaging leads to a decrease of the magnitude of the measured 3hJ(NC') constants, which varies locally (larger for -sheets, smaller for -helix). Two different spin-spin coupling mechanisms were identified. While mechanism I transmits spin polarization via an electric field effect, mechanism II involves also electron delocalization from the lone pair of the carbonyl oxygen to the antibonding orbital of the N-H bond. Mechanism I is more important in the crystal structure of ubiquitin, while in aqueous solution, mechanism II plays a larger role. It is possible to set up simple relationships between the spin-spin coupling constants associated with the H bond in proteins and the geometrical features of these bonds. The importance of the 3hJ(NC') and 1J(N-H) constants as descriptors for the H-bond is emphasized.

  20. Minimalist Relativistic Force Field: Prediction of Proton-Proton Coupling Constants in (1)H NMR Spectra Is Perfected with NBO Hybridization Parameters.

    Science.gov (United States)

    Kutateladze, Andrei G; Mukhina, Olga A

    2015-05-15

    We previously developed a reliable method for multiparametric scaling of Fermi contacts to achieve fast and accurate prediction of proton-proton spin-spin coupling constants (SSCC) in (1)H NMR. We now report that utilization of NBO hybridization coefficients for carbon atoms in the involved C-H bonds allows for a significant simplification of this parametric scheme, requiring only four general types of SSCCs: geminal, vicinal, 1,3-, and long-range constants. The method is optimized for inexpensive B3LYP/6-31G(d) molecular geometries. A new DU8 basis set, based on a training set of 475 experimental spin-spin coupling constants, is developed for hydrogen and common non-hydrogen atoms (Li, B, C, N, O, F, Si, P, S, Cl, Se, Br, I) to calculate Fermi contacts. On a test set of 919 SSCCs from a diverse collection of natural products and complex synthetic molecules the method gave excellent accuracy of 0.29 Hz (rmsd) with the maximum unsigned error not exceeding 1 Hz.

  1. Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

    Science.gov (United States)

    Zubrienė, Asta; Matulienė, Jurgita; Baranauskienė, Lina; Jachno, Jelena; Torresan, Jolanta; Michailovienė, Vilma; Cimmperman, Piotras; Matulis, Daumantas

    2009-01-01

    The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding. PMID:19582223

  2. Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

    Directory of Open Access Journals (Sweden)

    Vilma Michailovienė

    2009-06-01

    Full Text Available The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC and thermal shift assay (TSA is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90aN and the binding of ethoxzolamide to human carbonic anhydrase (hCAII were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90aN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.

  3. Study of the effect hydrogen binding in the solvation of alkaline earth cations with MeOH in nitromethane using 1 H NMR technique and determination of ionic solvation number

    CERN Document Server

    Alizadeh, N

    2001-01-01

    A proton NMR method for the study of the effect hydrogen binding and determination of solvation numbers of alkaline earth cations with methanol (MeOH) in in tromethane (NM) as diluent is described. The method is based on monitoring the resonance frequency of MeOH protons as a function of MeOH to metal ion mole ratio at constant metal ion concentration. the average solvation number of cation, n, at any MeOH/ metal ion mole ration was calculated from the NMR chemical shift-mole ration data and was plotted against the mole ration values. The solvation numbers of alkaline earth cations were obtained from the limiting values of the corresponding n, vs. mole ratio plots.

  4. 13C-detected NMR experiments for measuring chemical shifts and coupling constants in nucleic acid bases.

    Science.gov (United States)

    Fiala, Radovan; Sklenár, Vladimír

    2007-10-01

    The paper presents a set of two-dimensional experiments that utilize direct (13)C detection to provide proton-carbon, carbon-carbon and carbon-nitrogen correlations in the bases of nucleic acids. The set includes a (13)C-detected proton-carbon correlation experiment for the measurement of (13)C-(13)C couplings, the CaCb experiment for correlating two quaternary carbons, the HCaCb experiment for the (13)C-(13)C correlations in cases where one of the carbons has a proton attached, the HCC-TOCSY experiment for correlating a proton with a network of coupled carbons, and a (13)C-detected (13)C-(15)N correlation experiment for detecting the nitrogen nuclei that cannot be detected via protons. The IPAP procedure is used for extracting the carbon-carbon couplings and/or carbon decoupling in the direct dimension, while the S(3)E procedure is preferred in the indirect dimension of the carbon-nitrogen experiment to obtain the value of the coupling constant. The experiments supply accurate values of (13)C and (15)N chemical shifts and carbon-carbon and carbon-nitrogen coupling constants. These values can help to reveal structural features of nucleic acids either directly or via induced changes when the sample is dissolved in oriented media.

  5. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  6. Electrostatic interactions in the binding pathway of a transient protein complex studied by NMR and isothermal titration calorimetry.

    Science.gov (United States)

    Meneses, Erick; Mittermaier, Anthony

    2014-10-03

    Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr-Purcell-Meiboom-Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes.

  7. Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry*

    Science.gov (United States)

    Meneses, Erick; Mittermaier, Anthony

    2014-01-01

    Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr–Purcell–Meiboom–Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes. PMID:25122758

  8. Partition coefficient vs. binding constant: How best to assess molecular lipophilicity.

    Science.gov (United States)

    Cevc, Gregor

    2015-05-01

    Partition coefficient, P, is the preferred descriptor of molecular lipo- or hydrophilicity, and thus of relationships between a solute (S, e.g., a drug), a polar medium (W, e.g., an aqueous buffer), and an essentially apolar, organic, medium or a drug carrier (O). The coefficient is commonly identified with the linear ratio of solute quantities in the two media, P=nSO/nSW, even to characterise solute association with or binding to a surface (e.g., of a HPLC column or a drug carrier). To check the latter practice correctness-and credibility of the prevailing P definition-this paper compares an ideal solute distribution between two separate homogeneous fluid media (i.e., partitioning) to solute association with a uniform surface immersed in one such medium (i.e., binding). This reveals that solute partitioning and binding fundamentally differ and can only exceptionally be described, or analysed, with similar equations. Nonlinearised formulae that describe partitioning (Eq. (9)) and binding (Eq. (11)) can yield similar lipophilicity descriptor values only if solute preparation is relatively dilute; employing a large organic medium fraction is helpful in this respect. Additional prerequisites for partitioning and binding models match are: 1:1 stoichiometry at the association maximum and identical interfacial area of solute and organic medium molecules. If these requirements are not met, binding model yields different, potentially somewhat higher, but more often up to >10 times lower results than partitioning model. The main reason is saturation of organic medium with solute molecules. Partitioning model excludes this phenomenon, which cannot always be prevented by focussing on dilute solute preparations. The current practice of using a linear model and approximate equations to study partitioning or binding can cause large errors (>10(3)×), and is one possible reason for the notoriously high experimental logP values scattering. The latter makes logP predictions more

  9. Determination of ¹J(⁵⁹Co-⁵⁹Co) scalar coupling constants in the tetrahedral mixed-metal cluster HFeCo₃(CO)₁₀(PCyH₂)(PPh₂[CH₂C(O)Ph]) using COSY-type NMR experiments.

    Science.gov (United States)

    Kempgens, Pierre; Rosé, Jacky

    2011-03-01

    Two-dimensional ⁵⁹Co correlation spectroscopy (COSY) and double-quantum-filtered (DQF) COSY NMR experiments are reported for the tetrahedral mixed-metal cluster HFeCo₃(CO)₁₀(PCyH₂)(PPh₂[CH₂C(O)Ph]), which consists from the point of view of ⁵⁹Co NMR spectroscopy, of an AMX system of three-spin S=7/2. Both 2D NMR spectra prove the existence of a J scalar coupling constant between non-equivalent ⁵⁹Co nuclei. By contrast to what happens with the conventional 2D ⁵⁹Co COSY NMR spectrum, it was possible to simulate the 2D ⁵⁹Co DQF-COSY NMR spectrum by density matrix calculations in order to extract the values of the ¹J(⁵⁹Co-⁵⁹Co) coupling constants. The comparison between experimental and theoretical 2D NMR spectra gives spin-couplings constants of several hundreds Hertz for this cluster. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. NMR insights into the conformational properties of Man-9 and its recognition by two HIV binding proteins.

    Science.gov (United States)

    Shahzad-Ul-Hussan, Syed; Sastry, Mallika; Lemmin, Thomas; Soto, Cinque; Loesgen, Sandra; Scott, Danielle A; Davison, Jack Robert; O'Connor, Robert; Kwong, Peter D; Bewley, Carole A

    2017-02-06

    Man9GlcNAc2 (Man-9) present at the surface of HIV constitutes the binding sites of several HIV neutralizing agents and mammalian lectin DC-SIGN that is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in free state and when bound by an HIV entry inhibitor protein and define the minimum epitopes of two HIV binding proteins using NMR spectroscopy. In this regard we developed a robust expression system for production of 13C,15N-labeled glycans in mammalian cells that facilitated the implementation of 3D 13C-edited spectra to deconvolute spectral overlap allowing for the solution structure determination of Man-9. The studies reveal that Man-9 interacts with HIV-binding proteins via distinct binding epitopes and adopts divergent conformations in the bound state. In combination with molecular dynamics we find that these receptor-bound conformations are sampled by Man-9 in the free state suggesting a mechanism for diverse recognition.

  11. Absolute NMR shielding scales and nuclear spin-rotation constants in (175)LuX and (197)AuX (X = (19)F, (35)Cl, (79)Br and (127)I).

    Science.gov (United States)

    Demissie, Taye B; Jaszuński, Michał; Komorovsky, Stanislav; Repisky, Michal; Ruud, Kenneth

    2015-10-28

    We present nuclear spin-rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants, and shielding spans of all the nuclei in (175)LuX and (197)AuX (X = (19)F, (35)Cl, (79)Br, (127)I), calculated using coupled-cluster singles-and-doubles with a perturbative triples (CCSD(T)) correction theory, four-component relativistic density functional theory (relativistic DFT), and non-relativistic DFT. The total nuclear spin-rotation constants determined by adding the relativistic corrections obtained from DFT calculations to the CCSD(T) values are in general in agreement with available experimental data, indicating that the computational approach followed in this study allows us to predict reliable results for the unknown spin-rotation constants in these molecules. The total NMR absolute shielding constants are determined for all the nuclei following the same approach as that applied for the nuclear spin-rotation constants. In most of the molecules, relativistic effects significantly change the computed shielding constants, demonstrating that straightforward application of the non-relativistic formula relating the electronic contribution to the nuclear spin-rotation constants and the paramagnetic contribution to the shielding constants does not yield correct results. We also analyze the origin of the unusually large absolute shielding constant and its relativistic correction of gold in AuF compared to the other gold monohalides.

  12. Absolute NMR shielding scales and nuclear spin–rotation constants in {sup 175}LuX and {sup 197}AuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br and {sup 127}I)

    Energy Technology Data Exchange (ETDEWEB)

    Demissie, Taye B., E-mail: taye.b.demissie@uit.no; Komorovsky, Stanislav; Repisky, Michal; Ruud, Kenneth [Centre for Theoretical and Computational Chemistry, Department of Chemistry, UiT The Arctic University of Norway, N-9037 Tromsø (Norway); Jaszuński, Michał [Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44, 01 224 Warszawa (Poland)

    2015-10-28

    We present nuclear spin–rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants, and shielding spans of all the nuclei in {sup 175}LuX and {sup 197}AuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I), calculated using coupled-cluster singles-and-doubles with a perturbative triples (CCSD(T)) correction theory, four-component relativistic density functional theory (relativistic DFT), and non-relativistic DFT. The total nuclear spin–rotation constants determined by adding the relativistic corrections obtained from DFT calculations to the CCSD(T) values are in general in agreement with available experimental data, indicating that the computational approach followed in this study allows us to predict reliable results for the unknown spin–rotation constants in these molecules. The total NMR absolute shielding constants are determined for all the nuclei following the same approach as that applied for the nuclear spin–rotation constants. In most of the molecules, relativistic effects significantly change the computed shielding constants, demonstrating that straightforward application of the non-relativistic formula relating the electronic contribution to the nuclear spin–rotation constants and the paramagnetic contribution to the shielding constants does not yield correct results. We also analyze the origin of the unusually large absolute shielding constant and its relativistic correction of gold in AuF compared to the other gold monohalides.

  13. Absolute NMR shielding scales and nuclear spin-rotation constants in 175LuX and 197AuX (X = 19F, 35Cl, 79Br and 127I)

    Science.gov (United States)

    Demissie, Taye B.; Jaszuński, Michał; Komorovsky, Stanislav; Repisky, Michal; Ruud, Kenneth

    2015-10-01

    We present nuclear spin-rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants, and shielding spans of all the nuclei in 175LuX and 197AuX (X = 19F, 35Cl, 79Br, 127I), calculated using coupled-cluster singles-and-doubles with a perturbative triples (CCSD(T)) correction theory, four-component relativistic density functional theory (relativistic DFT), and non-relativistic DFT. The total nuclear spin-rotation constants determined by adding the relativistic corrections obtained from DFT calculations to the CCSD(T) values are in general in agreement with available experimental data, indicating that the computational approach followed in this study allows us to predict reliable results for the unknown spin-rotation constants in these molecules. The total NMR absolute shielding constants are determined for all the nuclei following the same approach as that applied for the nuclear spin-rotation constants. In most of the molecules, relativistic effects significantly change the computed shielding constants, demonstrating that straightforward application of the non-relativistic formula relating the electronic contribution to the nuclear spin-rotation constants and the paramagnetic contribution to the shielding constants does not yield correct results. We also analyze the origin of the unusually large absolute shielding constant and its relativistic correction of gold in AuF compared to the other gold monohalides.

  14. Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS

    Energy Technology Data Exchange (ETDEWEB)

    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)

    2016-01-21

    Establishing connectivity and proximity of nuclei is an important step in elucidating the structure and dynamics of molecules in solids using magic angle spinning (MAS) NMR spectroscopy. Although recent studies have successfully demonstrated the feasibility of proton-detected multidimensional solid-state NMR experiments under ultrafast-MAS frequencies and obtaining high-resolution spectral lines of protons, assignment of proton resonances is a major challenge. In this study, we first re-visit and demonstrate the feasibility of 2D constant-time uniform-sign cross-peak correlation (CTUC-COSY) NMR experiment on rigid solids under ultrafast-MAS conditions, where the sensitivity of the experiment is enhanced by the reduced spin-spin relaxation rate and the use of low radio-frequency power for heteronuclear decoupling during the evolution intervals of the pulse sequence. In addition, we experimentally demonstrate the performance of a proton-detected pulse sequence to obtain a 3D {sup 1}H/{sup 13}C/{sup 1}H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t{sub 1} and t{sub 3} periods, respectively. In addition to through-space and through-bond {sup 13}C/{sup 1}H and {sup 13}C/{sup 13}C chemical shift correlations, the 3D {sup 1}H/{sup 13}C/{sup 1}H experiment also provides a COSY-type {sup 1}H/{sup 1}H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices ({sup 1}H/{sup 1}H chemical shift correlation spectrum) at different {sup 13}C chemical shift frequencies from the 3D {sup 1}H/{sup 13}C/{sup 1}H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the

  15. Constant-time 2D and 3D through-bond correlation NMR spectroscopy of solids under 60 kHz MAS.

    Science.gov (United States)

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy

    2016-01-21

    Establishing connectivity and proximity of nuclei is an important step in elucidating the structure and dynamics of molecules in solids using magic angle spinning (MAS) NMR spectroscopy. Although recent studies have successfully demonstrated the feasibility of proton-detected multidimensional solid-state NMR experiments under ultrafast-MAS frequencies and obtaining high-resolution spectral lines of protons, assignment of proton resonances is a major challenge. In this study, we first re-visit and demonstrate the feasibility of 2D constant-time uniform-sign cross-peak correlation (CTUC-COSY) NMR experiment on rigid solids under ultrafast-MAS conditions, where the sensitivity of the experiment is enhanced by the reduced spin-spin relaxation rate and the use of low radio-frequency power for heteronuclear decoupling during the evolution intervals of the pulse sequence. In addition, we experimentally demonstrate the performance of a proton-detected pulse sequence to obtain a 3D (1)H/(13)C/(1)H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t1 and t3 periods, respectively. In addition to through-space and through-bond (13)C/(1)H and (13)C/(13)C chemical shift correlations, the 3D (1)H/(13)C/(1)H experiment also provides a COSY-type (1)H/(1)H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices ((1)H/(1)H chemical shift correlation spectrum) at different (13)C chemical shift frequencies from the 3D (1)H/(13)C/(1)H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the 3D (1)H/(13)C/(1)H experiment would be useful to study the structure and dynamics of

  16. Polymer binding to carbon nanotubes in aqueous dispersions: residence time on the nanotube surface as obtained by NMR diffusometry.

    Science.gov (United States)

    Frise, Anton E; Pagès, Guilhem; Shtein, Michael; Pri Bar, Ilan; Regev, Oren; Furó, István

    2012-03-08

    The binding of block copolymer Pluronic F-127 in aqueous dispersions of single- (SWCNT) and multiwalled (MWCNT) carbon nanotubes has been studied by pulsed-field-gradient (PFG) (1)H NMR spectroscopy. We show that a major fraction of polymers exist as a free species while a minor fraction is bound to the carbon nanotubes (CNT). The polymers exchange between these two states with residence times on the nanotube surface of 24 ± 5 ms for SWCNT and of 54 ± 11 ms for MWCNT. The CNT concentration in the solution was determined by improved thermal gravimetric analysis (TGA) indicating that the concentration of SWCNT dispersed by F-127 was significantly higher than that for MWCNT. For SWCNT, the area per adsorbed Pluronic F-127 molecule is estimated to be about 40 nm(2).

  17. Dielectric constant of graphene-on-polarized substrate: A tight-binding model study

    Indian Academy of Sciences (India)

    SIVABRATA SAHU; S K S PARASHAR; G C ROUT

    2017-07-01

    We report here a microscopic tight-binding theoretical study of the dynamic dielectric response of graphene-on-polarizable substrate with impurity. The Hamiltonian consists of first, second and third nearest neighbour electron hopping interactions besides doping and substrate-induced effects on graphene. We have introduced electron–electron correlation effect at A and B sublattices of graphene which is considered within Hartree–Fock mean-field approximation. The electron occupancies at both sublattices are calculated and solvedself-consistently and numerically for both up- and down-spin orientations. The polarization function appearing in the dielectric function is a two-particle Green’s function which is calculated by using Zubarev’s Green’s function technique. The temperature and optical frequency-dependent dielectric function is evaluated and compared with experimental data by varying Coulomb correlation energy, substrate-induced gap and impurity concentrations.

  18. Studies of DNA-binding properties of lafutidine as adjuvant anticancer agent to calf thymus DNA using multi-spectroscopic approaches, NMR relaxation data, molecular docking and dynamical simulation.

    Science.gov (United States)

    Yang, Hongqin; Tang, Peixiao; Tang, Bin; Huang, Yanmei; He, Jiawei; Li, Shanshan; Li, Hui

    2017-06-01

    The interactions between lafutidine (LAF) and calf thymus DNA (ctDNA) have been investigated both experimentally and theoretically. UV-vis absorption studies confirmed that LAF binds to ctDNA through non-covalent interactions. Fluorescence quenching and time-resolved fluorescence spectroscopy studies showed that the binding of LAF with ctDNA occurred through static quenching mechanism, resulting in the formation of a LAF-ctDNA complex. The binding constants (K) of the complex were found to be around 10(3)M(-1) via NMR relaxation rates and fluorescence data, and the calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played major roles in the binding of LAF to ctDNA. The changes in CD spectra indicated that LAF induced a slight perturbation on the base stacking and helicity of B-DNA. A comparative study of the LAF-ctDNA complex with respect to potassium iodide quenching experiments and competition displacement assays with ethidium bromide, acridine orange, and Hoechst 33258 probes suggested that LAF interacted with ctDNA by minor groove mode. Molecular docking analysis further supported the minor groove binding. Molecular dynamics simulation indicated that LAF depart from the C-G region of DNA, but it can steadily bind with the middle part of DNA composed by A-T base pairs. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. NMR studies of a new family of DNA binding proteins: the THAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gervais, Virginie, E-mail: virginie.gervais@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France); Campagne, Sebastien [ETH Zurich (Switzerland); Durand, Jade; Muller, Isabelle; Milon, Alain, E-mail: alain.milon@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France)

    2013-05-15

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  20. USING 19F-NMR SPECTROSCOPY TO DETERMINE TRIFLURALIN BINDING TO SOIL

    Science.gov (United States)

    Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soil-bound trifluralin residues, 14

  1. Predicting the Strength of Anion-π Interactions of Substituted Benzenes: the Development of Anion-π Binding Substituent Constants.

    Science.gov (United States)

    Bagwill, Christina; Anderson, Christa; Sullivan, Elizabeth; Manohara, Varun; Murthy, Prithvi; Kirkpatrick, Charles C; Stalcup, Apryll; Lewis, Michael

    2016-11-23

    A computational study aimed at accurately predicting the strength of the anion-π binding of substituted benzenes is presented. The anion-π binding energies (Ebind) of 37 substituted benzenes and the parent benzene, with chloride or bromide were investigated at the MP2(full)/6-311++G** level of theory. In addition, energy decomposition analysis was performed on 27 selected chloride-arene complexes via symmetry adapted perturbation theory (SAPT), using the SAPT2+ approach. Initial efforts aimed to correlate the anion-π Ebind values with the sum of the Hammett constants σp (Σσp) or σm (Σσm), as done by others. This proved a decent approach for predicting the binding strength of aromatics with electron-withdrawing substituents. For the Cl(-)-substituted benzene Ebind values, the correlation with the Σσp and Σσm values of aromatics with electron-withdrawing groups had r(2) values of 0.89 and 0.87 respectively. For the Br(-)-substituted benzene Ebind values, the correlation with the Σσp and Σσm values of aromatics with electron-withdrawing groups had r(2) values of 0.90 and 0.87. However, adding aromatics with electron-donating substituents to the investigation caused the correlation to deteriorate. For the Cl(-)-substituted benzene complexes the correlation between Ebind values and the Hammett constants had r(2) = 0.81 for Σσp and r(2) = 0.84 for Σσm. For the Br(-)-substituted benzene complexes, the respective r(2) values were 0.71 for Σσp and 0.79 for Σσm. The deterioration in correlation upon consideration of substituted benzenes with electron-donating substituents is due to the anion-π binding energies becoming more attractive regardless of what type of substituent is added to the aromatic. A similar trend has been reported for parallel face-to-face substituted benzene-benzene binding. This is certainly counter to what electrostatic arguments would predict for trends in anion-π binding energies, and this discrepancy is further highlighted

  2. Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles.

    Science.gov (United States)

    Su, Xun-Cheng; McAndrew, Kerry; Huber, Thomas; Otting, Gottfried

    2008-02-06

    Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.

  3. Sulfoalkyl ether-alkyl ether cyclodextrin derivatives, their synthesis, NMR characterization, and binding of 6alpha-methylprednisolone.

    Science.gov (United States)

    Tongiani, Serena; Velde, David Vander; Ozeki, Tetsuya; Stella, Valentino J

    2005-11-01

    The objective of this study is to see if random alkyl ethers of various sulfoalkyl ether cyclodextrins can be synthesized and characterized. The purpose of the alkylation was to test the hypothesis that an increase in the "height" of a cyclodextrins cavity would help in the binding/complexation of larger more structurally complex molecules. The synthesis of new cyclodextrin derivatives comprising a mixture of sulfoalkyl ether and alkyl ether substituents on the same cyclodextrin ring was performed in aqueous alkaline solutions using various sultones and alkylsulfates. The method presented provided an easy and efficient way to modify cyclodextrins avoiding the use of organic solvents and high quantities of alkylating agents and could be carried out in either a two step or "one pot" single step process. Purification was by neutralization followed by ultrafiltration. The derivatives were characterized by 1D, ((1)H and (13)C), and a 2D NMR technique (HMQC, Heteronuclear Multiple Quantum Coherence). The combination of these techniques allowed an analysis of the degree of substitution and the site of substitution on the cyclodextrin (CD) nucleus. For both beta- and gamma-CD, sulfoakylation was preferred on the 2 > 3 > 6 hydroxyls while alkylation was preferred 6 > 2 > 3. Due to the simultaneous presence of short alkyl ether chains and negatively charged sulfoalkyl ether chains, these mixed water-soluble cyclodextrin derivatives, especially those of gamma-cyclodextrin, should be able to bind more complex drugs. The improved binding capacity of these new modified CDs with the model drug 6alpha-methylprednisolone is reported.

  4. Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding

    Science.gov (United States)

    Viegas, Aldino; Manso, Joao; Nobrega, Franklin L.; Cabrita, Eurico J.

    2011-01-01

    Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor…

  5. Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding

    Science.gov (United States)

    Viegas, Aldino; Manso, Joao; Nobrega, Franklin L.; Cabrita, Eurico J.

    2011-01-01

    Saturation transfer difference (STD) NMR has emerged as one of the most popular ligand-based NMR techniques for the study of protein-ligand interactions. The success of this technique is a consequence of its robustness and the fact that it is focused on the signals of the ligand, without any need of processing NMR information about the receptor…

  6. Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

    Science.gov (United States)

    Canales, Angeles; Rodríguez-Salarichs, Javier; Trigili, Chiara; Nieto, Lidia; Coderch, Claire; Andreu, José Manuel; Paterson, Ian; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2011-08-19

    The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

  7. Comparative study of binding constants from Love wave surface acoustic wave and surface plasmon resonance biosensors using kinetic analysis.

    Science.gov (United States)

    Lee, Sangdae; Kim, Yong-Il; Kim, Ki-Bok

    2013-11-01

    Biosensors are used in a variety of fields for early diagnosis of diseases, measurement of toxic contaminants, quick detection of pathogens, and separation of specific proteins or DNA. In this study, we fabricated and evaluated the capability of a high sensitivity Love wave surface acoustic wave (SAW) biosensor. The experimental setup was composed of the fabricated 155-MHz Love wave SAW biosensor, a signal measurement system, a liquid flow system, and a temperature-control system. Subsequently, we measured the lower limit of detection (LOD) of the 155-MHz Love wave SAW biosensor, and calculated the association and dissociation constants between protein G and anti-mouse IgG using kinetic analysis. We compared these results with those obtained using a commercial surface plasmon resonance (SPR) biosensor. We found that the LOD of the SAW biosensor for anti-mouse IgG and mouse IgG was 0.5 and 1 microg/ml, respectively, and the resultant equilibrium association and dissociation constants were similar to the corresponding values obtaining using the commercial SPR biosensor. Thus, we conclude that the fabricated 155-MHz Love wave SAW biosensor exhibited the high sensitivity of the commercial SPR biosensor and was able to analyze the binding properties of the ligand and receptor by kinetic analysis similarly to the commercial SPR biosensor.

  8. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

    Directory of Open Access Journals (Sweden)

    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  9. NMR characterization of the DNA binding properties of a novel Hoechst 33258 analogue peptide building block

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; Behrens, Carsten; Jacobsen, Jens Peter

    2002-01-01

    A novel aryl-bis-benzimidazole amino acid analogue of the DNA-binding compound Hoechst 33258 has recently been designed for incorporation in peptide combinatorial libraries by replacing the N-methylpiperazine group with a carboxyl group and the hydroxy group with an amino-methyl group. The DNA...... preference with the bis-benzimidazole moiety displaced toward the 3'-end from the center of the duplex. Two families of models of the complexes with A(5) and A(3)T(3) were derived with restrained molecular dynamics based on a large set of 70 and 61, respectively, intermolecular ligand NOEs. Both models give...... a picture of a tightly fitting ligand with close van der Waals contacts with the walls of the minor groove and with the two benzimidazole and the amide hydrogens involved in bifurcated cross-strand hydrogen bonds to adenine N3 and thymine O2. The minor groove width of the models correlate well...

  10. NMR chemical shielding and spin-spin coupling constants of liquid NH3: a systematic investigation using the sequential QM/MM method.

    Science.gov (United States)

    Gester, Rodrigo M; Georg, Herbert C; Canuto, Sylvio; Caputo, M Cristina; Provasi, Patricio F

    2009-12-31

    The NMR spin coupling parameters, (1)J(N,H) and (2)J(H,H), and the chemical shielding, sigma((15)N), of liquid ammonia are studied from a combined and sequential QM/MM methodology. Monte Carlo simulations are performed to generate statistically uncorrelated configurations that are submitted to density functional theory calculations. Two different Lennard-Jones potentials are used in the liquid simulations. Electronic polarization is included in these two potentials via an iterative procedure with and without geometry relaxation, and the influence on the calculated properties are analyzed. B3LYP/aug-cc-pVTZ-J calculations were used to compute the (1)J(N,H) constants in the interval of -67.8 to -63.9 Hz, depending on the theoretical model used. These can be compared with the experimental results of -61.6 Hz. For the (2)J(H,H) coupling the theoretical results vary between -10.6 to -13.01 Hz. The indirect experimental result derived from partially deuterated liquid is -11.1 Hz. Inclusion of explicit hydrogen bonded molecules gives a small but important contribution. The vapor-to-liquid shifts are also considered. This shift is calculated to be negligible for (1)J(N,H) in agreement with experiment. This is rationalized as a cancellation of the geometry relaxation and pure solvent effects. For the chemical shielding, sigma((15)N) calculations at the B3LYP/aug-pcS-3 show that the vapor-to-liquid chemical shift requires the explicit use of solvent molecules. Considering only one ammonia molecule in an electrostatic embedding gives a wrong sign for the chemical shift that is corrected only with the use of explicit additional molecules. The best result calculated for the vapor to liquid chemical shift Delta sigma((15)N) is -25.2 ppm, in good agreement with the experimental value of -22.6 ppm.

  11. NMR spectra and potentiometry studies of aluminum(III) binding with coenzyme NAD+ in acidic aqueous solutions.

    Science.gov (United States)

    Yang, Xiaodi; Bi, Shuping; Yang, Xiaoliang; Yang, Li; Hu, Jun; Liu, Jian; Yang, Zhengbiao

    2003-06-01

    Complexation and conformational studies of coenzyme NAD+ with aluminum were conducted in acidic aqueous solutions (pH 2-5) by means of potentiometry as well as multinuclear (1H, 13C, 31P, 27Al) and two-dimensional (1H, 1H-NOESY) NMR spectroscopy. These led to the following results: (1) Al could coordinate with NAD+ through the following binding sites: N7' of adenine and pyrophosphate free oxygen (O(A)1, O(N)1,O(A)2) to form various mononuclear 1:1 (AlLH23+, AlLH2+) and 2:1 (AlL2-) species, and dinuclear 2:2 (Al2L22+) species. (2) The conformations of NAD+ and Al-NAD+ depended on the solvents and different species in the complexes. The results suggest the occurrence of an Al-linked complexation, which causes structural changes at the primary recognition sites and secondary conformational alterations for coenzymes. This finding will help us to understand role of Al in biological enzyme reaction systems.

  12. Characterization of Sodium Mobility and Binding by (23) Na NMR Spectroscopy in a Model Lipoproteic Emulsion Gel for Sodium Reduction.

    Science.gov (United States)

    Okada, Kyle S; Lee, Youngsoo

    2017-07-01

    The effects of formulation and processing parameters on sodium availability in a model lipid/protein-based emulsion gel were studied for purposes of sodium reduction. Heat-set model gels were prepared with varying levels of protein, lipid, and NaCl contents and high pressure homogenization treatments. Single quantum and double quantum-filtered (23) Na NMR spectroscopy experiments were used to characterize sodium mobility, structural order around "bound" (restricted mobility) sodium, and sodium binding, which have been correlated to saltiness perception in food systems previously. Total sodium mobility was lower in gels with higher protein or fat content, and was not affected by changes in homogenization pressure. The gels with increased protein, fat, or homogenization pressure had increased structure surrounding "bound" sodium and more relative "bound" sodium due to increased interfacial protein interactions. The data obtained in this study provide information on factors affecting sodium availability, which can be applied towards sodium reduction in lipid/protein-based foods. © 2017 Institute of Food Technologists®.

  13. Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

    Science.gov (United States)

    Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

    2016-04-14

    NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

  14. Analyzing protein-ligand interactions by dynamic NMR spectroscopy.

    Science.gov (United States)

    Mittermaier, Anthony; Meneses, Erick

    2013-01-01

    Nuclear magnetic resonance (NMR) spectroscopy can provide detailed information on protein-ligand interactions that is inaccessible using other biophysical techniques. This chapter focuses on NMR-based approaches for extracting affinity and rate constants for weakly binding transient protein complexes with lifetimes of less than about a second. Several pulse sequences and analytical techniques are discussed, including line-shape simulations, spin-echo relaxation dispersion methods (CPMG), and magnetization exchange (EXSY) experiments.

  15. (S)Pinning down protein interactions by NMR

    DEFF Research Database (Denmark)

    Teilum, Kaare; Kunze, Micha Ben Achim; Erlendsson, Simon

    2017-01-01

    all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative...... descriptions of protein interactions. In this review we provide an easy-access approach to NMR for the non-NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background...... and illustrate simple protein-ligand interactions and as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction...

  16. DFT study of NH{sub 3} adsorption on the (5,0), (8,0), (5,5) and (6,6) single-walled carbon nanotubes. Calculated binding energies, NMR and NQR parameters

    Energy Technology Data Exchange (ETDEWEB)

    Shirvani, Bahram B.; Beheshtian, Javad; Esrafili, Mehdi D. [Department of Chemistry, Tarbiat Modares University, P.O. Box: 14115-175, Tehran (Iran, Islamic Republic of); Hadipour, Nasser L., E-mail: hadipour@modares.ac.i [Department of Chemistry, Tarbiat Modares University, P.O. Box: 14115-175, Tehran (Iran, Islamic Republic of)

    2010-03-15

    Behavior of a single NH{sub 3} molecule adsorbed on external surface of H-capped (5,5), (6,6), (5,0), and (8,0) single-walled carbon nanotubes (SWCNTs) is studied via DFT calculations. Binding energies clearly exhibit adsorption dependence on tube diameter. {sup 13}C, {sup 15}N and {sup 1}H chemical shielding tensors are calculated at the B3LYP level using GIAO method. NMR calculations reveal that {sup 13}C chemical shielding of (8,0) is more sensitive to NH{sub 3} adsorption compared to (5,5), (6,6) and (5,0) tubes. {sup 15}N and {sup 1}H chemical shielding correlate noticeably with diameter of the nanotubes. {sup 14}N and {sup 2}H nuclear quadrupole coupling constants, C{sub Q}, and asymmetry parameter, eta, reveal the remarkable effect of NH{sub 3} adsorption on electronic structure of the SWCNTs.

  17. CORCEMA refinement of the bound ligand conformation within the protein binding pocket in reversibly forming weak complexes using STD-NMR intensities

    Science.gov (United States)

    Jayalakshmi, V.; Rama Krishna, N.

    2004-05-01

    We describe an intensity-restrained optimization procedure for refining approximate structures of ligands within the protein binding pockets using STD-NMR intensity data on reversibly forming weak complexes. In this approach, the global minimum for the bound-ligand conformation is obtained by a hybrid structure refinement method involving CORCEMA calculation of intensities and simulated annealing optimization of torsion angles of the bound ligand using STD-NMR intensities as experimental constraints and the NOE R-factor as the pseudo-energy function to be minimized. This method is illustrated using simulated STD data sets for typical carbohydrate and peptide ligands. Our procedure also allows for the optimization of side chain torsion angles of protein residues within the binding pocket. This procedure is useful in refining and improving initial models based on crystallography or computer docking or other algorithms to generate models for the bound ligand (e.g., a lead compound) within the protein binding pocket compatible with solution STD-NMR data. This method may facilitate structure-based drug design efforts.

  18. /sup 1/H and /sup 31/P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites

    Energy Technology Data Exchange (ETDEWEB)

    Scott, E.V.; Jones, R.L.; Banville, D.L.; Zon, G.; Marzilli, L.G.; Wilson, W.D.

    1988-02-09

    Imino proton and /sup 31/P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of /sup 1/H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different /sup 1/H and /sup 31/P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. Both upfield and downfield shifts were seen for imino proton signals for base pairs adjacent to ActD binding sites. This suggests that ActD has considerable long-range effects on oligomer conformation. Anticooperativity was also seen in NMR studies with a second class of oligomers containing alternating GC sequences longer than GCGC. It was found that in any three consecutive GC binding sites only two ActD can be bound. Anticooperativity can, thus, define the apparent number of base pairs in the binding site of a intercalator. Binding results with poly(d(G-C)) x poly(d(G-C)) were similar to those obtained with an oligomer containing four consecutive GC sites.

  19. NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1985-08-13

    Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual

  20. Flexible Acyclic Polyol-Chloride Anion Complexes and Their Characterization by Photoelectron Spectroscopy and Variable Temperature Binding Constant Determinations

    Energy Technology Data Exchange (ETDEWEB)

    Shokri, Alireza; Wang, Xue B.; Wang, Yangping; O' Doherty, George A.; Kass, Steven R.

    2016-03-17

    Flexible acyclic alcohols with 1–5 hydroxyl groups were bound to chloride anion and these complexes were interrogated by negative ion photoelectron spectroscopy and companion density functional theory computations. The resulting vertical detachment energies are reproduced on average to 0.10 eV by M06-2X/aug-cc-pVTZ predictions and range from 4.45 – 5.96 eV. These values are 0.84 – 2.35 eV larger than the adiabatic detachment energy of Cl– as a result of the larger hydrogen bond networks in the bigger polyols. Adiabatic detachment energies of the alcohol–Cl– clusters are more difficult to determine both experimentally and computationally. This is due to the large geometry changes that occur upon photodetachment and the large bond dissociation energy of H–Cl which enables the resulting chlorine atom to abstract a hydrogen from any of the methylene (CH2) or methine (CH) positions. Both ionic and non-ionic hydrogen bonds (i.e., OH•••Cl– and OH•••OH•••Cl–) form in the larger polyols complexes, and are found to be energetically comparable. Subtle structural differences, consequently can lead to the formation of different types of hydrogen bonds and maximizing the ionic ones is not always preferred. Solution equilibrium binding constants between the alcohols and tetrrabuylammonium chloride (TBACl) in acetonitrile at -24.2, 22.0, and 53.6 °C were also determined. The free energies of association are nearly identical for all of the substrates (i.e., ΔG° = -2.8 ± 0.7 kcal mol–1). Compensating enthalpy and entropy values reveal, contrary to expectation and the intrinsic gas-phase preferences, that the bigger systems with more hydroxyl groups are entropically favored and enthalpically disfavored relative to the smaller species. This suggests that more solvent molecules are released upon binding TBACl to alcohols with more hydroxyl groups and is consistent with the measured negative heat capacities. These quantities increase with

  1. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, M.; Kuby, S.A.; Mildvan A.S.

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme, appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase, with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of ..beta..-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% ..cap alpha..-helix, 38% ..beta..-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possible due to disorder, it can be fit by using methods developed on well-characterized globular proteins. The CD spectrum is best fit by assuming the presence of at most 13% ..cap alpha..-helix in the peptide, 24 +/- 2% ..beta..-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformation changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assess by CD.

  2. Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Aki, Kenzo; Tohyama, Yumi; Harano, Yuichi; Kawakami, Toru; Saito, Hiroyuki; Okamura, Emiko

    2017-04-15

    Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells. By introducing 4-trifluoromethyl-l-phenylalanine to the N terminus of R8, the non-endocytic membrane translocation of (19)F-labeled R8 ((19)F-R8) into a human myeloid leukemia cell line was observed at 4 °C with a time resolution in the order of minutes. (19)F NMR successfully detected real-time R8 translocation: the binding to anionic GAGs at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. The NMR concentration analysis enabled quantification of how much of R8 was staying in the respective translocation processes with time in situ. Taken together, our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of CPPs in cell membranes.

  3. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

    Science.gov (United States)

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

    2016-02-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  4. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

    2016-02-15

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  5. Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies.

    Science.gov (United States)

    Yang, Hongqin; Huang, Yanmei; Liu, Jiuyang; Tang, Peixiao; Sun, Qiaomei; Xiong, Xinnuo; Tang, Bin; He, Jiawei; Li, Hui

    2017-09-11

    Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

  6. Binding events of (S )-N -(3-oxo-octanoyl)-homoserine lactone with agrobacterium tumefaciens mutant cells studied by saturation transfer difference NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cabeca, Luis Fernando; Pomini, Armando Mateus; Cruz, Pedro Luiz R.; Marsaioli, Anita J. [University of Campinas (UNICAMP), SP (Brazil). Chemistry Inst.

    2011-07-01

    Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of b-cyclodextrin as acyl-HSL carrier were also provided. (author)

  7. NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

    Science.gov (United States)

    Yang, Yinshan; Boze, Hélène; Chemardin, Patrick; Padilla, André; Moulin, Guy; Tassanakajon, Anchalee; Pugnière, Martine; Roquet, Françoise; Destoumieux-Garzón, Delphine; Gueguen, Yannick; Bachère, Evelyne; Aumelas, André

    2009-03-01

    The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs.

  8. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    Science.gov (United States)

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  9. Binding modes of phosphonic acid derivatives adsorbed on TiO2 surfaces: Assignments of experimental IR and NMR spectra based on DFT/PBC calculations

    Science.gov (United States)

    Geldof, D.; Tassi, M.; Carleer, R.; Adriaensens, P.; Roevens, A.; Meynen, V.; Blockhuys, F.

    2017-01-01

    A DFT study on the adsorption of a series of phosphonic acids (PAs) on the TiO2 anatase (101) and (001) surfaces was performed. The adsorption energies and geometries of the most stable binding modes were compared to literature data and the effect of the inclusion of dispersion forces in the energy calculations was gauged. As the (101) surface is the most exposed surface of TiO2 anatase, the calculated chemical shifts and vibrational frequencies of PAs adsorbed on this surface were compared to experimental 31P and 17O NMR and IR data in order to assign the two possible binding modes (mono- and bidentate) to peaks and bands in these spectra; due to the corrugated nature of anatase (101) tridentate binding is not possible on this surface. Analysis of the calculated and experimental 31P chemical shifts indicates that both monodentate and bidentate binding modes are present. For the reactive (001) surface, the results of the calculations indicate that both bi- and tridentate binding modes result in stable systems. Due to the particular sensitivity of 17O chemical shifts to hydrogen bonding and solvent effects, the model used is insufficient to assign these spectra at present. Comparison of calculated and experimental IR spectra leads to the conclusion that IR spectroscopy is not suitable for the characterization of the different binding modes of the adsorption complexes.

  10. TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.

    Science.gov (United States)

    Kalverda, Arnout P; Gowdy, James; Thompson, Gary S; Homans, Steve W; Henderson, Peter J F; Patching, Simon G

    2014-06-01

    Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

  11. Molecular statics calculations of proton binding to goethite surfaces: A new approach to estimation of stability constants for multisite surface complexation models

    Science.gov (United States)

    Rustad, James R.; Felmy, Andrew R.; Hay, Benjamin P.

    1996-05-01

    A new approach to estimating stability constants for proton binding in multisite surface complexation models is presented. The method is based on molecular statics computation of energies for the formation of proton vacancies and interstitials in ideal periodic slabs representing the (100), (110), (010), (001), and (021) surfaces of goethite. Gas-phase energies of clusters representing the hydrolysis products of ferric iron are calculated using the same potential energy functions used for the surface. These energies are linearly related to the hydrolysis constants for ferric iron in aqueous solution. Stability constants for proton binding at goethite surfaces are estimated by assuming the same log K- Δ E relationship for goethite surface protonation reactions. These stability constants predict a pH of zero charge of 8.9, in adequate agreement with measurements on CO 2-free goethite. The estimated stability constants differ significantly from previous estimations based on Pauling bond strength. We find that nearly all the surface oxide ions are reactive; nineteen of the twenty-six surface sites investigated have log Kint between 7.7 and 9.4. This implies a site density between fifteen and sixteen reactive sites/nm for crystals dominated by (110) and (021) crystal faces.

  12. A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Belousova, Lubov; Plesner, Igor

    1993-01-01

    The quantitative aspects of mitochondrial creatinekinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the...

  13. Ab initio and relativistic DFT study of spin-rotation and NMR shielding constants in XF6 molecules, X = S, Se, Te, Mo, and W

    Science.gov (United States)

    Ruud, Kenneth; Demissie, Taye B.; Jaszuński, Michał

    2014-05-01

    We present an analysis of the spin-rotation and absolute shielding constants of XF6 molecules (X = S, Se, Te, Mo, W) based on ab initio coupled cluster and four-component relativistic density-functional theory (DFT) calculations. The results show that the relativistic contributions to the spin-rotation and shielding constants are large both for the heavy elements as well as for the fluorine nuclei. In most cases, incorporating the computed relativistic corrections significantly improves the agreement between our results and the well-established experimental values for the isotropic spin-rotation constants and their anisotropic components. This suggests that also for the other molecules, for which accurate and reliable experimental data are not available, reliable values of spin-rotation and absolute shielding constants were determined combining ab initio and relativistic DFT calculations. For the heavy nuclei, the breakdown of the relationship between the spin-rotation constant and the paramagnetic contribution to the shielding constant, due to relativistic effects, causes a significant error in the total absolute shielding constants.

  14. Ab initio and relativistic DFT study of spin–rotation and NMR shielding constants in XF{sub 6} molecules, X = S, Se, Te, Mo, and W

    Energy Technology Data Exchange (ETDEWEB)

    Ruud, Kenneth [Centre for Theoretical and Computational Chemistry, Department of Chemistry, University of Tromsø—The Arctic University of Norway, N-9037 Tromsø (Norway); Demissie, Taye B. [Centre for Theoretical and Computational Chemistry, Department of Chemistry, University of Tromsø—The Arctic University of Norway, N-9037 Tromsø (Norway); Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warszawa, Kasprzaka 44 (Poland); Jaszuński, Michał, E-mail: michal.jaszunski@icho.edu.pl [Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warszawa, Kasprzaka 44 (Poland)

    2014-05-21

    We present an analysis of the spin–rotation and absolute shielding constants of XF{sub 6} molecules (X = S, Se, Te, Mo, W) based on ab initio coupled cluster and four-component relativistic density-functional theory (DFT) calculations. The results show that the relativistic contributions to the spin–rotation and shielding constants are large both for the heavy elements as well as for the fluorine nuclei. In most cases, incorporating the computed relativistic corrections significantly improves the agreement between our results and the well-established experimental values for the isotropic spin–rotation constants and their anisotropic components. This suggests that also for the other molecules, for which accurate and reliable experimental data are not available, reliable values of spin–rotation and absolute shielding constants were determined combining ab initio and relativistic DFT calculations. For the heavy nuclei, the breakdown of the relationship between the spin–rotation constant and the paramagnetic contribution to the shielding constant, due to relativistic effects, causes a significant error in the total absolute shielding constants.

  15. Binding constants of inclusion complexes of nitroimidazoles with {beta}-cyclodextrins in the absence and presence of PVP

    Energy Technology Data Exchange (ETDEWEB)

    Chadha, Renu [University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014 (India)]. E-mail: renukchadha2004@yahoo.co.in; Jain, D.V.S. [Depatment of Chemistry, Panjab University, Chandigarh 160014 (India); Aggarwal, Amit [University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014 (India); Singh, Surjit [GND University, Amritsar, Panjab (India); Thakur, Deepika [University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014 (India)

    2007-07-01

    Thermodynamics of complexation of 5-nitroimidazoles with {beta}-cyclodextrin and its methylated and hydroxypropyl derivatives in water and in 0.25% polyvinylpyrrolidone are determined by solution calorimetry. A 1:1 stoichiometry was established. The equilibrium constant (K) for all the nitroimidazoles fall in the range 1000-1900 M{sup -1} suitable for use of cyclodextrins as drug carriers. The complexation ability is significantly enhanced by methylation of the {beta}-cyclodextrin. The stability constant increased in the order metronidazole < ornidazole < tinidazole < secnidazole. The presence of polyvinylpyrrolidone enhances the stability constants.

  16. Hyperfine coupling constant for {sup 59}Co estimated from a high-field susceptibility and high-field NMR shift in ferromagnetic Co{sub 2}TiGa and Co{sub 2}VGa

    Energy Technology Data Exchange (ETDEWEB)

    Nishihara, H; Furutani, Y; Wada, T [Faculty of Science and Technology, Ryukoku University, Otsu 520-2194 (Japan); Kanomata, T [Faculty of Engineering, Tohoku Gakuin University, Tagajo 985-8537 (Japan); Kobayashi, K; Ishida, K [Graduate School of Engineering, Tohoku University, Sendai 980-8579 (Japan); Kainuma, R [Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577 (Japan); Koyama, K; Watanabe, K, E-mail: nishihara@rins.ryukoku.ac.j [Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2010-01-01

    The hyperfine coupling constant for {sup 59}Co in the ferromagnetic state of a Heusler alloy Co{sub 2}TiGa in the high-field range has been estimated to be 170 kOe per 1 {mu}{sub B} of the moment of cobalt atom from the high-field magnetic susceptibility of 2.3 x 10{sup -6} emu/gOe measured at 5 K in a field range from 20 to 70 kOe and a reported positive high-field NMR shift of +0.83 percent. The value of the hyperfine coupling constant has been found to be even larger in the case of Co{sub 2}VGa. These features suggest that the positive hyperfine field at Co nucleus in the Co-based Heusler alloys is due to conduction electron polarization rather than a transferred hyperfine field.

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

    Energy Technology Data Exchange (ETDEWEB)

    Live, D.H.; Cowburn, D.

    1987-10-06

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

  18. N-acetylglyoxylic amide bearing a nitrophenyl group as anion receptors: NMR and X-ray investigations on anion binding and selectivity

    Science.gov (United States)

    Suryanti, Venty; Bhadbhade, Mohan; Black, David StC; Kumar, Naresh

    2017-10-01

    N-Nitrophenylglyoxylic amides 1 and 2 in presence of tetrabutylammonium cation (TBA) act as receptors for anions HSO4-, Cl-, Br- and NO3- as investigated by NMR studies. The receptors formed 1:1 host-guest complexes in solution. X-ray structure of 1 along with TBA that bind a chloride anion is reported. Molecule 1 showed the highest selectivity for HSO4- anion over others measured. X-ray structure of the bound Cl- revealed a pocket containing the anion making strong (Nsbnd H⋯Cl) and weak hydrogen bonds (Csbnd H⋯Cl) that contribute to the recognition of the chloride anion. Nsbnd H and Csbnd H hydrogen bonds resulted in a relatively strong binding for chloride ions.

  19. Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants.

    Science.gov (United States)

    Han, Yong-Woon; Matsumoto, Tomoko; Yokota, Hiroaki; Kashiwazaki, Gengo; Morinaga, Hironobu; Hashiya, Kaori; Bando, Toshikazu; Harada, Yoshie; Sugiyama, Hiroshi

    2012-12-01

    N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

  20. A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

    Science.gov (United States)

    Andrási, Melinda; Lehoczki, Gábor; Nagy, Zoltán; Gyémánt, Gyöngyi; Pungor, András; Gáspár, Attila

    2015-06-01

    This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. An introduction to biological NMR spectroscopy.

    Science.gov (United States)

    Marion, Dominique

    2013-11-01

    NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP).

  2. NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

    Science.gov (United States)

    Koutmou, Kristin S.; Casiano-Negroni, Anette; Getz, Melissa M.; Pazicni, Samuel; Andrews, Andrew J.; Penner-Hahn, James E.; Al-Hashimi, Hashim M.; Fierke, Carol A.

    2010-01-01

    Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn2+ bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn2+ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new “metal cocktail” chemical shift perturbation strategy involving titrations with , Zn2+, and confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution. PMID:20133747

  3. Solution structure of the 45-residue ATP-binding peptide of adenylate kinase as determined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, E.M.; Kuby, S.A.; Mildyan, A.S.

    1986-05-01

    In the X-ray structure of adenylate kinase residues 1-45 exist as 47% ..cap alpha..-helix, 29% ..beta..-structure (strands and turns) and 24% coil. The solution structure of a synthetic peptide corresponding to residues 1-45, which constitutes the MgATP binding site was studied by 3 independent spectroscopic methods. Globularity of the peptide was shown by its broad NMR resonances which narrow upon denaturation, and by its ability to bind MgATP with similar affinity and conformation as the intact enzyme does. COSY and NOESY NMR methods at 250 and 500 MHz reveal proximities among NH, C..cap alpha.., and C..beta.. protons indicative of >20% ..cap alpha..-helix, and >20% ..beta..-structure. Correlation of regions of secondary structure with the primary sequence by 2D NMR indicates at least one ..cap alpha..-helix (res. 23 to 29) and two ..beta..-strands (res. 12 to 15 and 34 to 38). The broad amide I band in the deconvoluted FTIR spectrum could be fit as the sum of 4 peaks due to specific secondary structures, yielding less than or equal to=45% ..cap alpha..-helix, less than or equal to=40% ..beta..-structure and greater than or equal to=15% coil. The CD spectrum, from 185-250 nm, interpreted with a 3-parameter basis set, yielded 20 +/- 5% ..cap alpha..=helix, and less than or equal to=20% ..beta..-structure. The solution structure of peptide 1-45 thus approximates that of residues 1-45 in the crystal.

  4. Applications of a tight-binding total energy method for transition and noble metals Elastic Constants, Vacancies, and Surfaces of Monatomic Metals

    CERN Document Server

    Mehl, M J; Mehl, Michael J.; Papaconstantopoulos, Dimitrios A.

    1996-01-01

    A recent tight-binding scheme provides a method for extending the results of first principles calculations to regimes involving $10^2 - 10^3$ atoms in a unit cell. The method uses an analytic set of two-center, non-orthogonal tight-binding parameters, on-site terms which change with the local environment, and no pair potential. The free parameters in this method are chosen to simultaneously fit band structures and total energies from a set of first-principles calculations for monatomic fcc and bcc crystals. To check the accuracy of this method we evaluate structural energy differences, elastic constants, vacancy formation energies, and surface energies, comparing to first-principles calculations and experiment. In most cases there is good agreement between this theory and experiment. We present a detailed account of the method, a complete set of tight-binding parameters, and results for twenty-nine of the alkaline earth, transition and noble metals.

  5. In depth analysis on the binding sites of adamantane derivatives in HCV (hepatitis C virus p7 channel based on the NMR structure.

    Directory of Open Access Journals (Sweden)

    Qi-Shi Du

    Full Text Available BACKGROUND: The recently solved solution structure of HCV (hepatitis C virus p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine was determined in a hydrophobic pocket. However the pharmocophore (-NH2 of the ligand was not assigned a specific binding site. RESULTS: The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T/6-311+G(d,p and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed. CONCLUSIONS: Some useful viewpoints are concluded as follows. (1 The amino group (-NH2 of adamantane derivatives is protonated (-NH3+, and the positively charged cation may form cation-π interactions with aromatic amino acids. (2 The aromatic amino acids (His17, Phe20, and Trp21 are the possible binding sites for the protonated amino group (-NH3+ of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3 The higher inhibition potent of rimantadine than amantadine probably because of its higher pKa value (pKa = 10.40 and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed.

  6. Indirect spin-spin coupling constants in CH{sub 4}, SiH{sub 4} and GeH{sub 4} - Gas-phase NMR experiment and ab initio calculations

    Energy Technology Data Exchange (ETDEWEB)

    Antusek, Andrej [Slovak University of Technology in Bratislava, Faculty of Materials Science and Technology in Trnava, Paulinska 16, 917 24 Trnava (Slovakia); Department of Chemistry, Nicolaus Copernicus University, Gagarina 7, PL 87-100 Torun (Poland); Kedziera, Dariusz [Department of Chemistry, Nicolaus Copernicus University, Gagarina 7, PL 87-100 Torun (Poland); Jackowski, Karol [Laboratory of NMR Spectroscopy, Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw (Poland); Jaszunski, Michal [Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44, 01224 Warsaw (Poland)], E-mail: michaljz@icho.edu.pl; Makulski, Wlodzimierz [Laboratory of NMR Spectroscopy, Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw (Poland)

    2008-09-03

    New values of the indirect spin-spin coupling constants in CH{sub 4}, SiH{sub 4} and GeH{sub 4}, derived from experiment and ab initio calculations, are reported. The new experimental values of {sup 1}J(CH), {sup 1}J(SiH) and {sup 1}J(GeH) are obtained from gas-phase NMR spectra. The dependence of the measured one-bond coupling constants on the density is analysed and the results are extrapolated to zero-density point to eliminate the effects due to intermolecular forces. In the calculation of the coupling constants, at the nonrelativistic level coupled cluster singles and doubles (CCSD) perturbation theory is used and the basis set convergence of the results is discussed. The relativistic corrections are estimated from Dirac-Hartree-Fock (DHF) calculations. The final theoretical values are obtained adding available estimates of the vibrational and temperature corrections. The agreement of the calculated and experimental {sup 1}J(XH), X = C, Si, Ge, constants is very satisfying, the differences are approximately 1-3%.

  7. Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

    Science.gov (United States)

    Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

    2012-01-01

    An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

  8. (19) F NMR Spectroscopic Analysis of the Binding Modes in Triple-Helical Peptide Nucleic Acid (PNA)/MicroRNA Complexes.

    Science.gov (United States)

    Tähtinen, Ville; Granqvist, Lotta; Murtola, Merita; Strömberg, Roger; Virta, Pasi

    2017-05-23

    Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of (19) F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by (19) F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. NMR studies of metalloproteins.

    Science.gov (United States)

    Li, Hongyan; Sun, Hongzhe

    2012-01-01

    Metalloproteins represent a large share of the proteomes, with the intrinsic metal ions providing catalytic, regulatory, and structural roles critical to protein functions. Structural characterization of metalloproteins and identification of metal coordination features including numbers and types of ligands and metal-ligand geometry, and mapping the structural and dynamic changes upon metal binding are significant for understanding biological functions of metalloproteins. NMR spectroscopy has long been used as an invaluable tool for structure and dynamic studies of macromolecules. Here we focus on the application of NMR spectroscopy in characterization of metalloproteins, including structural studies and identification of metal coordination spheres by hetero-/homo-nuclear metal NMR spectroscopy. Paramagnetic NMR as well as (13)C directly detected protonless NMR spectroscopy will also be addressed for application to paramagnetic metalloproteins. Moreover, these techniques offer great potential for studies of other non-metal binding macromolecules.

  10. Localized molecular orbital analysis of the effect of electron correlation on the anomalous isotope effect in the NMR spin-spin coupling constant in methane

    DEFF Research Database (Denmark)

    Zarycz, M. Natalia C.; Sauer, Stephan P. A.; Provasi, Patricio F.

    2014-01-01

    We discuss the effect of electron correlation on the unexpected differential sensitivity (UDS) in the 1J(C-H) coupling constant of CH4 using a decomposition into contributions from localized molecular orbitals and compare with the 1J(N-H) coupling constant in NH3. In particular we discuss the well...... known fact that uncorrelated coupled Hartree-Fock (CHF) calculations are not able to reproduce the UDS in methane. For this purpose we have implemented for the first time a localized molecular orbital analysis for the second order polarization propagator approximation with coupled cluster singles...... and doubles amplitudes - SOPPA(CCSD) in the DALTON program. Comparing the changes in the localized orbital contributions at the correlated SOPPA and SOPPA(CCSD) levels and at the uncorrelated CHF level, we find that the latter overestimates the effect of stretching the bond between the coupled atoms...

  11. The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies.

    Science.gov (United States)

    Wang, Tuo; Chen, Yuning; Tabuchi, Akira; Cosgrove, Daniel J; Hong, Mei

    2016-12-01

    The wall-loosening actions of β-expansins are known primarily from studies of EXPB1 extracted from maize (Zea mays) pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state nuclear magnetic resonance experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing (13)C-enriched maize CWs with EXPB1 functionalized with a Mn(2+) tag, we measured Mn(2+)-induced PRE Strong (1)H and (13)C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly substituted GAX and GAX of low substitution, which binds cellulose. This study provides molecular evidence of β-expansin's target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.

  12. The potential utility of predicted one bond carbon-proton coupling constants in the structure elucidation of small organic molecules by NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Chandrasekhar Venkata

    Full Text Available NMR spectroscopy is the most popular technique used for structure elucidation of small organic molecules in solution, but incorrect structures are regularly reported. One-bond proton-carbon J-couplings provide additional information about chemical structure because they are determined by different features of molecular structure than are proton and carbon chemical shifts. However, these couplings are not routinely used to validate proposed structures because few software tools exist to predict them. This study assesses the accuracy of Density Functional Theory for predicting them using 396 published experimental observations from a diverse range of small organic molecules. With the B3LYP functional and the TZVP basis set, Density Functional Theory calculations using the open-source software package NWChem can predict one-bond CH J-couplings with good accuracy for most classes of small organic molecule. The root-mean-square deviation after correction is 1.5 Hz for most sp3 CH pairs and 1.9 Hz for sp2 pairs; larger errors are observed for sp3 pairs with multiple electronegative substituents and for sp pairs. These results suggest that prediction of one-bond CH J-couplings by Density Functional Theory is sufficiently accurate for structure validation. This will be of particular use in strained ring systems and heterocycles which have characteristic couplings and which pose challenges for structure elucidation.

  13. The potential utility of predicted one bond carbon-proton coupling constants in the structure elucidation of small organic molecules by NMR spectroscopy.

    Science.gov (United States)

    Venkata, Chandrasekhar; Forster, Mark J; Howe, Peter W A; Steinbeck, Christoph

    2014-01-01

    NMR spectroscopy is the most popular technique used for structure elucidation of small organic molecules in solution, but incorrect structures are regularly reported. One-bond proton-carbon J-couplings provide additional information about chemical structure because they are determined by different features of molecular structure than are proton and carbon chemical shifts. However, these couplings are not routinely used to validate proposed structures because few software tools exist to predict them. This study assesses the accuracy of Density Functional Theory for predicting them using 396 published experimental observations from a diverse range of small organic molecules. With the B3LYP functional and the TZVP basis set, Density Functional Theory calculations using the open-source software package NWChem can predict one-bond CH J-couplings with good accuracy for most classes of small organic molecule. The root-mean-square deviation after correction is 1.5 Hz for most sp3 CH pairs and 1.9 Hz for sp2 pairs; larger errors are observed for sp3 pairs with multiple electronegative substituents and for sp pairs. These results suggest that prediction of one-bond CH J-couplings by Density Functional Theory is sufficiently accurate for structure validation. This will be of particular use in strained ring systems and heterocycles which have characteristic couplings and which pose challenges for structure elucidation.

  14. NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)]4.

    Science.gov (United States)

    Pradeep, Tarikere Palakshan; Barthwal, Ritu

    2016-01-01

    The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

  15. Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag

    Energy Technology Data Exchange (ETDEWEB)

    Sengupta, Ishita; Gao, Min; Arachchige, Rajith J.; Nadaud, Philippe S. [The Ohio State University, Department of Chemistry and Biochemistry (United States); Cunningham, Timothy F.; Saxena, Sunil [University of Pittsburgh, Department of Chemistry (United States); Schwieters, Charles D. [National Institutes of Health, Center for Information Technology (United States); Jaroniec, Christopher P., E-mail: jaroniec@chemistry.ohio-state.edu [The Ohio State University, Department of Chemistry and Biochemistry (United States)

    2015-01-15

    Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu{sup 2+} binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC–Cu{sup 2+} K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ∼10 Å of the Cu{sup 2+} center experience considerable transverse PREs leading to severely attenuated resonances in 2D {sup 15}N–{sup 13}C correlation spectra. For more distant residues, electron–nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for {sup 15}N–Cu{sup 2+} distances up to ∼20 Å.

  16. Ferrous Iron Binding Key to Mms6 Magnetite Biomineralisation: A Mechanistic Study to Understand Magnetite Formation Using pH Titration and NMR Spectroscopy.

    Science.gov (United States)

    Rawlings, Andrea E; Bramble, Jonathan P; Hounslow, Andrea M; Williamson, Michael P; Monnington, Amy E; Cooke, David J; Staniland, Sarah S

    2016-06-01

    Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. HypCal, a general-purpose computer program for the determination of standard reaction enthalpy and binding constant values by means of calorimetry.

    Science.gov (United States)

    Arena, Giuseppe; Gans, Peter; Sgarlata, Carmelo

    2016-09-01

    The program HypCal has been developed to provide a means for the simultaneous determination, from data obtained by isothermal titration calorimetry, of both standard enthalpy of reaction and binding constant values. The chemical system is defined in terms of species of given stoichiometry rather than in terms of binding models (e.g., independent or cooperative). The program does not impose any limits on the complexity of the chemical systems that can be treated, including competing ligand systems. Many titration curves may be treated simultaneously. HypCal can also be used as a simulation program when designing experiments. The use of the program is illustrated with data obtained with nicotinic acid (niacin, pyridine-3 carboxylic acid). Preliminary experiments were used to establish the rather different titration conditions for the two sets of titration curves that are needed to determine the parameters for protonation of the carboxylate and amine groups.

  18. Communication: Localized molecular orbital analysis of the effect of electron correlation on the anomalous isotope effect in the NMR spin-spin coupling constant in methane

    Energy Technology Data Exchange (ETDEWEB)

    Zarycz, M. Natalia C., E-mail: mnzarycz@gmail.com; Provasi, Patricio F., E-mail: patricio@unne.edu.ar [Department of Physics, University of Northeastern - CONICET, Av. Libertad 5500, Corrientes W3404AAS (Argentina); Sauer, Stephan P. A., E-mail: sauer@kiku.dk [Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø (Denmark)

    2014-10-21

    We discuss the effect of electron correlation on the unexpected differential sensitivity (UDS) in the {sup 1}J(C–H) coupling constant of CH{sub 4} using a decomposition into contributions from localized molecular orbitals and compare with the {sup 1}J(N–H) coupling constant in NH{sub 3}. In particular, we discuss the well known fact that uncorrelated coupled Hartree-Fock (CHF) calculations are not able to reproduce the UDS in methane. For this purpose we have implemented for the first time a localized molecular orbital analysis for the second order polarization propagator approximation with coupled cluster singles and doubles amplitudes—SOPPA(CCSD) in the DALTON program. Comparing the changes in the localized orbital contributions at the correlated SOPPA and SOPPA(CCSD) levels and at the uncorrelated CHF level, we find that the latter overestimates the effect of stretching the bond between the coupled atoms on the contribution to the coupling from the localized bonding orbital between these atoms. This disturbs the subtle balance between the molecular orbital contributions, which lead to the UDS in methane.

  19. Measurement of homonuclear magnetic dipole-dipole interactions in multiple 1/2-spin systems using constant-time DQ-DRENAR NMR.

    Science.gov (United States)

    Ren, Jinjun; Eckert, Hellmut

    2015-11-01

    A new pulse sequence entitled DQ-DRENAR (Double-Quantum based Dipolar Recoupling Effects Nuclear Alignment Reduction) was recently described for the quantitative measurement of magnetic dipole-dipole interactions in homonuclear spin-1/2 systems involving multiple nuclei. As described in the present manuscript, the efficiency and performance of this sequence can be significantly improved, if the measurement is done in the constant-time mode. We describe both the theoretical analysis of this method and its experimental validation of a number of crystalline model compounds, considering both symmetry-based and back-to-back (BABA) DQ-coherence excitation schemes. Based on the combination of theoretical analysis and experimental results we discuss the effect of experimental parameters such as the chemical shift anisotropy (CSA), the spinning rate, and the radio frequency field inhomogeneity upon its performance. Our results indicate that constant-time (CT-) DRENAR is a method of high efficiency and accuracy for compounds with multiple homonuclear spin systems with particular promise for the analysis of stronger-coupled and short T2 spin systems.

  20. Isothermal titration calorimetry for drug design: Precision of the enthalpy and binding constant measurements and comparison of the instruments.

    Science.gov (United States)

    Linkuvienė, Vaida; Krainer, Georg; Chen, Wen-Yih; Matulis, Daumantas

    2016-12-15

    Isothermal titration calorimetry (ITC) is one of the most robust label- and immobilization-free techniques used to measure protein - small molecule interactions in drug design for the simultaneous determination of the binding affinity (ΔG) and the enthalpy (ΔH), both of which are important parameters for structure-thermodynamics correlations. It is important to evaluate the precision of the method and of various ITC instrument models by performing a single well-characterized reaction. The binding between carbonic anhydrase II and acetazolamide was measured by four ITC instruments - PEAQ-ITC, iTC200, VP-ITC, and MCS-ITC and the standard deviation of ΔG and ΔH was determined. Furthermore, the limit of an approach to reduce the protein concentration was studied for a high-affinity reaction (Kd = 0.3 nM), too tight to be measured by direct (non-displacement) ITC. Chemical validation of the enthalpy measurements is discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Konuma, Tsuyoshi [Icahn School of Medicine at Mount Sinai, Department of Structural and Chemical Biology (United States); Harada, Erisa [Suntory Foundation for Life Sciences, Bioorganic Research Institute (Japan); Sugase, Kenji, E-mail: sugase@sunbor.or.jp, E-mail: sugase@moleng.kyoto-u.ac.jp [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)

    2015-12-15

    Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

  3. Determination of binding constants and stoichiometries of short-range, hydrogen-bonding solvation by use of a proton-transfer indicator reaction

    Science.gov (United States)

    Schullery, Stephen E.; Wojdyla, Stephen M.; Ostroski, Robert A.; Scott, Ronald M.

    1997-10-01

    A recent method for determination of stoichiometries and binding constants for short-range, hydrogen bonding solvation is reviewed and new results are presented. The method exploits the sensitivity of a proton-transfer equilibrium, KPT to changes in solvent composition. Solvation numbers and binding constants for primary and secondary stages of solvation of an aminephenol proton-transfer adduct and the phenol and amine are determined as adjustable parameters when model isotherms are fitted to KPT versus [ S] data, where [ S] is the concentration of a hydrogen-bonding minor component of a mixed solvent, Results for most of the twenty-three aprotic solvents investigated are modeled by bifurcation-type hydrogen bonding of two or more electron pairs to a single polar hydrogen. Results for the seven protic solvents studied, including new data for ethanol, 2-choloroethanol, and 2,2-dichloroethanol, are modeled by two, or possibly three, successive stages of solvation, assumed to involve hydrogen-bonded chains. Preliminary results indicate that solvation by water is amenable to this analysis.

  4. Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

    DEFF Research Database (Denmark)

    Erlendsson, Simon; Gotfryd, Kamil; Larsen, Flemming Hofmann

    2017-01-01

    The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been ...

  5. Proton NMR studies of (N-MeCys sup 3 ,N-MeCys sup 7 )TANDEM binding to DNA oligonucleotides: Sequence-specific binding at the TpA site

    Energy Technology Data Exchange (ETDEWEB)

    Addess, K.J.; Gilbert, D.E.; Feigon, J. (Univ. of California, Los Angeles (United States)); Olsen, R.K. (Utah State Univ., Logan (United States))

    1992-01-21

    (N-MeCys{sup 3},N-MeCys{sup 7})TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that (N-MeCys{sup 3},N-Me-Cys{sup 7})TANDEM binds strongly to DNA rich in A{center dot}T residues. However, it was not known whether specific binding of (N-MeCys{sup 3},N-MeCys{sup 7})TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers (d(GGATATCC)){sub 2} and (d(GGTTAACC)){sub 2}, (N-MeCys{sup 3},N-MeCys{sup 7})TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer (d(GGAATTCC)){sub 2} as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that (N-MeCys{sup 3},N-MeCys{sup 7})TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of (N-MeCys{sup 3},N-MeCys{sup 7})TANDEM binding to DNA are discussed.

  6. The effect of dielectric constant on binding energy and impurity self-polarization in a GaAs-Ga1- x Al x As spherical quantum dot

    Science.gov (United States)

    Mese, A. I.; Cicek, E.; Erdogan, I.; Akankan, O.; Akbas, H.

    2017-03-01

    The ground state, 1s, and the excited state, 2p, energies of a hydrogenic impurity in a GaAs-Ga1- x Al x As spherical quantum dot, are computed as a function of the donor positions. We study how the impurity self-polarization depends on the location of the impurity and the dielectric constant. The excited state anomalous impurity self-polarization in the quantum dot is found to be present in the absence of any external influence and strongly depends on the impurity position and the radius of the dot. Therefore, the excited state anomalous impurity self-polarization can give information about the impurity position in the system. Also, the variation of E_{b1s} and E_{b2p} with the dielectric constant can be utilized as a tool for finding out the correct dielectric constant of the dot material by measuring the 1s or 2p state binding energy for a fixed dot radius and a fixed impurity position.

  7. NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.

    Science.gov (United States)

    Kurniawan, N D; Atkins, A R; Bieri, S; Brown, C J; Brereton, I M; Kroon, P A; Smith, R

    2000-07-01

    The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

  8. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

    Science.gov (United States)

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-02-22

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

  9. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

    Science.gov (United States)

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-01-01

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation. PMID:28223697

  10. Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes.

    Science.gov (United States)

    Zelikin, Alexander N; Trukhanova, Elizabeth S; Putnam, David; Izumrudov, Vladimir A; Litmanovich, Andrey A

    2003-11-12

    The physicochemical characteristics of a nonviral gene delivery system will govern its functional bioactivity; however, empiricism dominates the literature in this field, and a significant deficiency of quantitative investigation and evaluation of nonviral gene delivery vehicles remains. Herein, we derive a physical model and experimental method to quantitatively determine the binding constants between a model polycationic nonviral gene delivery vehicle poly-L-histidine (PLH) and calf thymus DNA. The approach has utility to a variety of systems and is not limited to the described polymer model. The interaction of PLH with DNA was monitored by fluorescence quenching of an ethidium bromide probe in the pH range 4 to 8. The interaction increased with pH decrease with the most pronounced change between pH 6 and 7. The obtained pH-dependence of fraction of salt bonds formed between PLH and DNA was used to estimate pK(a) of PLH in the presence of DNA, which equaled 6.24. The interaction of PLH with DNA in the presence of added synthetic polyanions was studied by the same approach and found to be controlled by pH, nature of the charge groups of the polyanion, and its degree of polymerization. In the mixture with sodium poly(styrenesulfonate) the interaction was negligible in the whole studied pH range, whereas in the mixtures with sodium poly(acrylate) (PA) or sodium poly(methacrylate), DNA was able to compete effectively for the binding with PLH. For PA samples with degree of polymerization higher than degree of polymerization of PLH, DP(PA) > DP(PLH), the fraction of polycation bound to DNA was constant regardless of DP(PA.) In contrast, at DP(PA) PLH), a pronounced increase in the bound fraction was observed. It substantiates the notion that the binding energy of two polymers is mainly controlled by the DP of the shorter component of polyelectrolyte complex. The data on PLH distribution between DNA and added polyanion with different values of DP were treated according

  11. Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix.

    Science.gov (United States)

    Gershlak, Joshua R; Black, Lauren D

    2015-01-15

    We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for β1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the β1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the β1 integrin on cells grown on the adult ECM and found that the inhibition of β1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through β1 integrin binding to the complex composition. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Mathematical model for evaluating the Krebs cycle flux with non-constant glutamate-pool size by 13C-NMR spectroscopy. Evidence for the existence of two types of Krebs cycles in cells.

    Science.gov (United States)

    Tran-Dinh, S; Beganton, F; Nguyen, T T; Bouet, F; Herve, M

    1996-12-01

    A practical method using matrix operations is proposed for studying the isotopic transformation of glutamate, or any other metabolite isotopomers, in the Krebs cycle. Two mathematical models were constructed for evaluating the Krebs cycle flux where the enrichment of [2-13C]acetyl-CoA is not 100% and the total glutamate concentration remains constant or varies during incubation. A comparative study of [1-13C]glucose metabolism was subsequently carried out using Saccharomyces cerevisiae cells from two different strains (ATCC-9763 and NCYC-239) by 13C-NMR spectroscopy and biochemical techniques. The results show that there are two types of Krebs cycles in cells. The first is represented by the ATCC cells which contain a small amount of 2-oxoglutarate dehydrogenase and hence the flux in the Krebs cycle is negligible. With [1-13C]glucose as a carbon source, the 13C-NMR spectra of glutamate exhibit the C2 and C4 resonances that are almost equivalent and much greater than that of the C3. Labeled metabolites derived from [1-13C]glucose enter the Krebs cycle at two points: oxaloacetate and citrate. The second cell type is represented by NCYC-239. The C2 and C3 areas are equivalent and smaller than the C4 resonance. The results suggest that labeled metabolites enter the Krebs cycle only at the citrate level via acetyl-CoA, 2-oxoglutarate dehydrogenase is present but pyruvate carboxylase is virtually absent or inactivated. When both are incubated with glucose, the total concentration of glutamate was found to decrease with the incubation time. The fraction of glutamate in isotopic exchange with the Krebs cycle in NCYC-239 cells is about 2.6% and the reduction in glutamate concentration is about 0.5%/min. Using our model, with a variable glutamate pool size, good agreement between the theoretical and experimental data is obtained.

  13. Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation.

    Science.gov (United States)

    Getz, Melissa M; Andrews, Andy J; Fierke, Carol A; Al-Hashimi, Hashim M

    2007-02-01

    The P4 helix is an essential element of ribonuclease P (RNase P) that is believed to bind catalytically important metals. Here, we applied a combination of NMR residual dipolar couplings (RDCs) and a recently introduced domain-elongation strategy for measuring "motionally decoupled" relaxation data to characterize the structural dynamics of the P4 helix from Bacillus subtilis RNase P. In the absence of divalent ions, the two P4 helical domains undergo small amplitude (approximately 13 degrees) collective motions about an average interhelical angle of 10 degrees. The highly conserved U7 bulge and helical residue C8, which are proposed to be important for substrate recognition and metal binding, are locally mobile at pico- to nanosecond timescales and together form the pivot point for the collective domain motions. Chemical shift mapping reveals significant association of Mg2+ ions at the P4 major groove near the flexible pivot point at residues (A5, G22, G23) previously identified to bind catalytically important metals. The Mg2+ ions do not, however, significantly alter the structure or dynamics of P4. Analysis of results in the context of available X-ray structures of the RNA component of RNase P and structural models that include the pre-tRNA substrate suggest that the internal motions observed in P4 likely facilitate adaptive changes in conformation that take place during folding and substrate recognition, possibly aided by interactions with Mg2+ ions. Our results add to a growing view supporting the existence of functionally important internal motions in RNA occurring at nanosecond timescales.

  14. Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, A. Patricia [University of Washington, Department of Medicinal Chemistry, School of Pharmacy (United States); Spyracopoulos, Leo [Department of Biochemistry (Canada); Irvin, Randall T. [University of Alberta, Department of Medical Microbiology and Immunology (Canada); Sykes, Brian D. [Department of Biochemistry (Canada)

    2000-07-15

    The backbone dynamics of a {sup 15}N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys{sup 128}-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys{sup 144}) were probed by measurements of {sup 15}N NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The {sup 15}N longitudinal (T{sub 1}) and transverse (T{sub 2}) relaxation rates and the steady-state heteronuclear {l_brace}{sup 1}H{r_brace}-{sup 15}N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 deg. C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the 'model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153-162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II {beta}-turns spanning residues Asp{sup 134}-Glu-Gln-Phe{sup 137} and Pro{sup 139}-Lys-Gly-Cys{sup 142}, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

  15. Antigen-antibody selective recognition using LiTaO3 SH-SAW sensors: investigations on macromolecules effects on binding kinetic constants

    Science.gov (United States)

    Bergaoui, Y.; Zerrouki, C.; Fourati, N.; Fougnion, J. M.; Abdelghani, A.

    2011-10-01

    A gravimetric surface acoustic wave (SAW) biosensor, based on the biotin-streptavidin and antistreptavidin-streptavidin recognitions, has been carried out. A network analyser and a pulse excitation technique were used to monitor both amplitude and phase changes. The SAW biosensor presented a total selective recognition of streptavidin-antistreptavidin and HRPstreptavidin-antistreptavidin. The presence of HRP (Horseradish peroxidase) affects neither the selectivity nor the sensitivity (of order of 0.25°/nM) of the biosensor, nevertheless, it causes a reduction of binding kinetics by a factor ranging between 2 to 5, as well as a diminution of antistreptavidin saturation concentration (of 40%). Results showed that equilibrium constants can be different, depending on evaluation method (from saturation values or from linear part of the output signal variation according to solution concentration).

  16. Estimation of the pH-independent binding constants of alanylphenylalanine and leucylphenylalanine stereoisomers with beta-cyclodextrin in the presence of urea.

    Science.gov (United States)

    Li, J; Waldron, K C

    1999-01-01

    The separation of stereoisomers, particularly enantiomers, is important when their physiological activity differs. We have resolved the four stereoisomers each of alanylphenylalanine (Ala-Phe) and of leucylphenylalanine (Leu-Phe) by capillary electrophoresis using beta-cyclodextrin as a buffer additive and urea to enhance its solubility. A study of the influence of pH and beta-cyclodextrin concentration on the separations showed that weak inclusion complexes were formed between the dipeptides and chiral selector. It was found that pH could alter the migration order of enantiomers L-Ala-L-Phe and D-Ala-D-Phe, as well as L-Leu-L-Phe and D-Leu-D-Phe; however, there was no change in order for the other pairs of optical isomers. Electrophoretic mobility data were used to estimate the acid dissociation constants of the dipeptide isomers at pH < 7 with no chiral selector present. By varying the concentration of beta-cyclodextrin, the chiral selector, the binding constants of Ala-Phe and Leu-Phe optical isomers in their fully protonated and zwitterionic forms were estimated. For the four Ala-Phe stereoisomers, K = 42-66 M(-1) and 4-41 M(-1) for the cationic and zwitterionic forms, respectively. For the four Leu-Phe stereoisomers, K = 43-94 M(-1) and 1-28 M(-1) for the cationic and zwitterionic forms, respectively.

  17. ¹³C NMR-distance matrix descriptors: optimal abstract 3D space granularity for predicting estrogen binding.

    Science.gov (United States)

    Slavov, Svetoslav H; Geesaman, Elizabeth L; Pearce, Bruce A; Schnackenberg, Laura K; Buzatu, Dan A; Wilkes, Jon G; Beger, Richard D

    2012-07-23

    An improved three-dimensional quantitative spectral data-activity relationship (3D-QSDAR) methodology was used to build and validate models relating the activity of 130 estrogen receptor binders to specific structural features. In 3D-QSDAR, each compound is represented by a unique fingerprint constructed from (13)C chemical shift pairs and associated interatomic distances. Grids of different granularity can be used to partition the abstract fingerprint space into congruent "bins" for which the optimal size was previously unexplored. For this purpose, the endocrine disruptor knowledge base data were used to generate 50 3D-QSDAR models with bins ranging in size from 2 ppm × 2 ppm × 0.5 Å to 20 ppm × 20 ppm × 2.5 Å, each of which was validated using 100 training/test set partitions. Best average predictivity in terms of R(2)test was achieved at 10 ppm ×10 ppm × Z Å (Z = 0.5, ..., 2.5 Å). It was hypothesized that this optimum depends on the chemical shifts' estimation error (±4.13 ppm) and the precision of the calculated interatomic distances. The highest ranked bins from partial least-squares weights were found to be associated with structural features known to be essential for binding to the estrogen receptor.

  18. Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

    Energy Technology Data Exchange (ETDEWEB)

    Foster, Mark P.; Wuttke, Deborah S.; Clemens, Karen R.; Jahnke, Wolfgang; Radhakrishnan, Ishwar; Tennant, Linda; Reymond, Martine; Chung, John; Wright, Peter E. [Scripps Research Institute, Department of Molecular Biology and Skaggs Institute for Chemical Biology (United States)

    1998-07-15

    We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone{sup 1} H{sup N}, {sup 15}N,{sup 13} C{sup {alpha}} and{sup 13} C{sup {beta}} and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

  19. Elucidating connectivity and metal-binding structures of unlabeled paramagnetic complexes by 13C and 1H solid-state NMR under fast magic angle spinning.

    Science.gov (United States)

    Wickramasinghe, Nalinda P; Shaibat, Medhat A; Ishii, Yoshitaka

    2007-08-23

    Characterizing paramagnetic complexes in solids is an essential step toward understanding their molecular functions. However, methodologies to characterize chemical and electronic structures of paramagnetic systems at the molecular level have been notably limited, particularly for noncrystalline solids. We present an approach to obtain connectivities of chemical groups and metal-binding structures for unlabeled paramagnetic complexes by 13C and 1H high-resolution solid-state NMR (SSNMR) using very fast magic angle spinning (VFMAS, spinning speed >or=20 kHz). It is experimentally shown for unlabeled Cu(II)(Ala-Thr) that 2D 13C/1H correlation SSNMR under VFMAS provides the connectivity of chemical groups and assignments for the characterization of unlabeled paramagnetic systems in solids. We demonstrate that on the basis of the assignments provided by the VFMAS approach multiple 13C-metal distances can be simultaneously elucidated by a combination of measurements of 13C anisotropic hyperfine shifts and 13C T1 relaxation due to hyperfine interactions for this peptide-Cu(II) complex. It is also shown that an analysis of 1H anisotropic hyperfine shifts allows for the determination of electron-spin states in Fe(III)-chloroprotoporphyin-IX in solid states.

  20. 最高量子相关谱技术在同核偶合常数测定中的应用%MEASUREMENT OF HOMONUCLEAR COUPLING CONSTANTS USING TWO-DIMENSIONAL MAXIMUM-QUANTUM CORRELATION NMR SPECTROSCOPY (MAXY-NMR)

    Institute of Scientific and Technical Information of China (English)

    张许; 刘买利

    1999-01-01

    It has been a continuous interest in measurement of homonuclear scalar coupling constants using two-dimensional NMR spectroscopy because large chemical shift dispersions can efficiently increase spectral resolution. Numerous methods have been developed using homo- and hetero-nuclear correlation and successfully used for a variety of samples. Here we demonstrate an alternative approach based on maximum-quantum correlation NMR spectroscopy (MAXY NMR). The new method combines the advantages of two-dimensional chemical shift dispersion and the spectral editing feature of the MAXY approach and results in separated correlations of CH, CH2, and CH3 groups in a single experiment with enhanced chemical shift resolution. The method had been tested on a middle-sized molecule, dexamethasone, and a tridecapeptide, neurotensin.%偶合常数是一个重要的NMR参数,其数值与分子中化学键的二面角有关,可以为分子结构研究提供很重要的信息.多维NMR谱由于具有较大的化学位移分辨率,因此常常被用来测定同核或异核自旋-自旋偶合常数.本文介绍了利用最高量子相关技术(MAXY)测定同核偶合常数的方法.MAXY是最近发展的一种多维NMR谱编辑技术,可以使不同官能团(CH, CH2, CH3)的相关峰分布于不同的图谱区域,因此比常规的二维谱具有更高的化学位移分辨率.而且被分离开来的NMR相关峰呈吸收性线型,能清楚地展示各自的偶合分裂特征,可以直接用于测定偶合常数.

  1. Competitive binding exchange between alkali metal ions (K+, Rb+, and Cs+) and Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2: a 23Na and 1H NMR study.

    Science.gov (United States)

    Cesare Marincola, Flaminia; Virno, Ada; Randazzo, Antonio; Mocci, Francesca; Saba, Giuseppe; Lai, Adolfo

    2009-12-01

    A comparative study of the competitive cation exchange between the alkali metal ions K+, Rb+, and Cs+ and the Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2 was performed in aqueous solution by a combined use of the 23Na and 1H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G-quadruplex and, in particular, major differences in the behavior of Cs+ as compared to the other ions were found. Accordingly, Cs+ competes with Na+ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K+ and Rb+ are also able to replace sodium ions located inside the quadruplex. Furthermore, the 1H NMR results relative to the CsCl titration evidence a close approach of Cs+ ions to the phosphate groups in the narrow groove of [d(G4T4G4)]2. Based on a three-site exchange model, the 23Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs+ versus Na+ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G-quadruplex formed by 5'-guanosinemonophosphate and for the surface of double-helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA.

  2. The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

    DEFF Research Database (Denmark)

    Teilum, Kaare; Poulsen, F. M.; Akke, M.

    2006-01-01

    those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of k f and k u in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models...

  3. The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

    DEFF Research Database (Denmark)

    Teilum, Kaare; Poulsen, F. M.; Akke, M.

    2006-01-01

    those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of k f and k u in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models...

  4. Cooperative binding of midazolam with testosterone and alpha-naphthoflavone within the CYP3A4 active site: a NMR T1 paramagnetic relaxation study.

    Science.gov (United States)

    Cameron, Michael D; Wen, Bo; Allen, Kyle E; Roberts, Arthur G; Schuman, Jason T; Campbell, A Patricia; Kunze, Kent L; Nelson, Sidney D

    2005-11-01

    Recent studies have indicated that CYP3A4 exhibits non-Michaelis-Menten kinetics for numerous substrates. Both homo- and heterotropic activation have been reported, and kinetic models have suggested multiple substrates within the active site. We provide some of the first physicochemical data supporting the hypothesis of allosteric substrate binding within the CYP3A4 active site. Midazolam (MDZ) is metabolized by CYP3A4 to two hydroxylated metabolites, 1'- and 4-hydroxymidazolam. Incubations using purified CYP3A4 and MDZ showed that both alpha-naphthoflavone (alpha-NF) and testosterone affect the ratio of formation rates of 1'- and 4-hydroxymidazolam. Similar to previous reports, alpha-NF was found to promote formation of 1'-hydroxymidazolam, while testosterone stimulated formation of 4-hydroxymidazolam. NMR was used to measure the closest approach of individual MDZ protons to the paramagnetic heme iron of CYP3A4 using paramagnetic T(1) relaxation measurements. Solutions of 0.2 microM CYP3A4 with 500 microM MDZ resulted in calculated distances between 7.4 and 8.3 A for all monitored MDZ protons. The distances were statistically equivalent for all protons except C3-H and were consistent with the rotation within the active site or sliding parallel to the heme plane. When 50 microM alpha-NF was added, proton-heme iron distances ranged from 7.3 to 10.0 A. Consistent with kinetics of activation, the 1' position was situated closest to the heme, while the fluorophenyl 5-H proton was the furthest. Proton-heme iron distances for MDZ with CYP3A4 and 50 microM testosterone ranged from 7.7 to 9.0 A, with the flourophenyl 5-H proton furthest from the heme iron and the C4-H closest to the heme, also consistent with kinetic observations. When titrated with CYP3A4 in the presence of MDZ, testosterone and alpha-NF resonances themselves exhibited significant broadening and enhanced relaxation rates, indicating that these effector molecules were also bound within the CYP3A4 active

  5. Structure and equilibria of Ca 2+-complexes of glucose and sorbitol from multinuclear ( 1H, 13C and 43Ca) NMR measurements supplemented with molecular modelling calculations

    Science.gov (United States)

    Pallagi, A.; Dudás, Cs.; Csendes, Z.; Forgó, P.; Pálinkó, I.; Sipos, P.

    2011-05-01

    Ca 2+-complexation of D-glucose and D-sorbitol have been investigated with the aid of multinuclear ( 1H, 13C and 43Ca) NMR spectroscopy and ab initio quantum chemical calculations. Formation constants of the forming 1:1 complexes have been estimated from one-dimensional 13C NMR spectra obtained at constant ionic strength (1 M NaCl). Binding sites were identified from 2D 1H- 43Ca NMR spectra. 2D NMR measurements and ab initio calculations indicated that Ca 2+ ions were bound in a tridentate manner via the glycosidic OH, the ethereal oxygen in the ring and the OH on the terminal carbon for the α- and β-anomers of glucose and for sorbitol simultaneous binding of four hydroxide moieties (C1, C2, C4 and C6) was suggested.

  6. The prion protein binds thiamine.

    Science.gov (United States)

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.

  7. Macrocycles Containing Tin. The Preparation of Macrobicyclic Lewis Acidic Hosts Containing Two Tin Atoms and 119-Sn NMR Studies of Their Chloride and Bromide Binding Properties in Solution

    Science.gov (United States)

    1989-05-24

    the X-ray crystal structure of the chloro derivative 2a. 7 1,9-Diphenyl-1,9-distannabicyclo[7.7.7] tricosane (Sb) was purified by reverse phase...obtained in 85% yield; mp 116-119 0C; 13C NMR (CDCl3): 6 31.6,24.8,19.9; 119Sn NMR (CDCI3 ): 6 148.5. 1,9-Dichloro-1,9-distannabicyclo[7.7.7] tricosane

  8. De novo design of chiral organotin cancer drug candidates: validation of enantiopreferential binding to molecular target DNA and 5'-GMP by UV-visible, fluorescence, (1)H and (31)P NMR.

    Science.gov (United States)

    Arjmand, Farukh; Sharma, Girish Chandra; Sayeed, Fatima; Muddassir, Mohd; Tabassum, Sartaj

    2011-12-02

    N,N-bis[(R-/S-)-1-benzyl-2-ethoxyethane] tin (IV) complexes were synthesized by applying de novo design strategy by the condensation reaction of (R-/S-)2-amino-2-phenylethanol and dibromoethane in presence of dimethyltin dichloride and thoroughly characterized by elemental analysis, conductivity measurements, IR, ESI-MS, (1)H, (13)C and (119)Sn, multinuclear NMR spectroscopy and XRD study. Enantioselective and specific binding profile of R-enantiomer 1 in comparison to S-enantiomer 2 with ultimate molecular target CT-DNA was validated by UV-visible, fluorescence, circular dichroism, (1)H and (31)P NMR techniques. This was further corroborated well by interaction of 1 and 2 with 5'-GMP. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. NMR studies of metalloproteins

    OpenAIRE

    Li, H; H. Sun

    2011-01-01

    Metalloproteins represent a large share of the proteomes, with the intrinsic metal ions providing catalytic, regulatory, and structural roles critical to protein functions. Structural characterization of metalloproteins and identification of metal coordination features including numbers and types of ligands and metal-ligand geometry, and mapping the structural and dynamic changes upon metal binding are significant for understanding biological functions of metalloproteins. NMR spectroscopy has...

  10. β-NMR sample optimization

    CERN Document Server

    Zakoucka, Eva

    2013-01-01

    During my summer student programme I was working on sample optimization for a new β-NMR project at the ISOLDE facility. The β-NMR technique is well-established in solid-state physics and just recently it is being introduced for applications in biochemistry and life sciences. The β-NMR collaboration will be applying for beam time to the INTC committee in September for three nuclei: Cu, Zn and Mg. Sample optimization for Mg was already performed last year during the summer student programme. Therefore sample optimization for Cu and Zn had to be completed as well for the project proposal. My part in the project was to perform thorough literature research on techniques studying Cu and Zn complexes in native conditions, search for relevant binding candidates for Cu and Zn applicable for ß-NMR and eventually evaluate selected binding candidates using UV-VIS spectrometry.

  11. What Is the True Color of Fresh Meat? A Biophysical Undergraduate Laboratory Experiment Investigating the Effects of Ligand Binding on Myoglobin Using Optical, EPR, and NMR Spectroscopy

    Science.gov (United States)

    Linenberger, Kimberly; Bretz, Stacey Lowery; Crowder, Michael W.; McCarrick, Robert; Lorigan, Gary A.; Tierney, David L.

    2011-01-01

    With an increased focus on integrated upper-level laboratories, we present an experiment integrating concepts from inorganic, biological, and physical chemistry content areas. Students investigate the effects of ligand strength on the spectroscopic properties of the heme center in myoglobin using UV-vis, [superscript 1]H NMR, and EPR…

  12. What Is the True Color of Fresh Meat? A Biophysical Undergraduate Laboratory Experiment Investigating the Effects of Ligand Binding on Myoglobin Using Optical, EPR, and NMR Spectroscopy

    Science.gov (United States)

    Linenberger, Kimberly; Bretz, Stacey Lowery; Crowder, Michael W.; McCarrick, Robert; Lorigan, Gary A.; Tierney, David L.

    2011-01-01

    With an increased focus on integrated upper-level laboratories, we present an experiment integrating concepts from inorganic, biological, and physical chemistry content areas. Students investigate the effects of ligand strength on the spectroscopic properties of the heme center in myoglobin using UV-vis, [superscript 1]H NMR, and EPR…

  13. Enzyme dynamics from NMR spectroscopy.

    Science.gov (United States)

    Palmer, Arthur G

    2015-02-17

    CONSPECTUS: Biological activities of enzymes, including regulation or coordination of mechanistic stages preceding or following the chemical step, may depend upon kinetic or equilibrium changes in protein conformations. Exchange of more open or flexible conformational states with more closed or constrained states can influence inhibition, allosteric regulation, substrate recognition, formation of the Michaelis complex, side reactions, and product release. NMR spectroscopy has long been applied to the study of conformational dynamic processes in enzymes because these phenomena can be characterized over multiple time scales with atomic site resolution. Laboratory-frame spin-relaxation measurements, sensitive to reorientational motions on picosecond-nanosecond time scales, and rotating-frame relaxation-dispersion measurements, sensitive to chemical exchange processes on microsecond-millisecond time scales, provide information on both conformational distributions and kinetics. This Account reviews NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic (Escherichia coli) and thermophilic (Thermus thermophilus) bacteria, E. coli AlkB, and Saccharomyces cerevisiae triosephosphate isomerase to illustrate the contributions of conformational flexibility and dynamics to diverse steps in enzyme mechanism. Spin relaxation measurements and molecular dynamics (MD) simulations of the bacterial ribonuclease H enzymes show that the handle region, one of three loop regions that interact with substrates, interconverts between two conformations. Comparison of these conformations with the structure of the complex between Homo sapiens ribonuclease H and a DNA:RNA substrate suggests that the more closed state is inhibitory to binding. The large population of the closed conformation in T. thermophilus ribonuclease H contributes to the increased Michaelis constant compared with the E. coli enzyme. NMR spin relaxation and fluorescence spectroscopy have characterized a

  14. Varying Constants

    CERN Document Server

    Damour, Thibault Marie Alban Guillaume

    2003-01-01

    We review some string-inspired theoretical models which incorporate a correlated spacetime variation of coupling constants while remaining naturally compatible both with phenomenological constraints coming from geochemical data (Oklo; Rhenium decay) and with present equivalence principle tests. Barring unnatural fine-tunings of parameters, a variation of the fine-structure constant as large as that recently ``observed'' by Webb et al. in quasar absorption spectra appears to be incompatible with these phenomenological constraints. Independently of any model, it is emphasized that the best experimental probe of varying constants are high-precision tests of the universality of free fall, such as MICROSCOPE and STEP. Recent claims by Bekenstein that fine-structure-constant variability does not imply detectable violations of the equivalence principle are shown to be untenable.

  15. Elucidation of the CCR1- and CCR5-binding modes of MIP-1α by application of an NMR spectra reconstruction method to the transferred cross-saturation experiments

    Energy Technology Data Exchange (ETDEWEB)

    Yoshiura, Chie; Ueda, Takumi; Kofuku, Yutaka; Matsumoto, Masahiko; Okude, Junya; Kondo, Keita; Shiraishi, Yutaro; Shimada, Ichio, E-mail: shimada@iw-nmr.f.u-tokyo.ac.jp [The University of Tokyo, Graduate School of Pharmaceutical Sciences (Japan)

    2015-12-15

    C–C chemokine receptor 1 (CCR1) and CCR5 are involved in various inflammation and immune responses, and regulate the progression of the autoimmune diseases differently. However, the number of residues identified at the binding interface was not sufficient to clarify the differences in the CCR1- and CCR5-binding modes to MIP-1α, because the NMR measurement time for CCR1 and CCR5 samples was limited to 24 h, due to their low stability. Here we applied a recently developed NMR spectra reconstruction method, Conservation of experimental data in ANAlysis of FOuRier, to the amide-directed transferred cross-saturation experiments of chemokine receptors, CCR1 and CCR5, embedded in lipid bilayers of the reconstituted high density lipoprotein, and MIP-1α. Our experiments revealed that the residues on the N-loop and β-sheets of MIP-1α are close to both CCR1 and CCR5, and those in the C-terminal helix region are close to CCR5. These results suggest that the genetic influence of the single nucleotide polymorphisms of MIP-1α that accompany substitution of residues in the C-terminal helix region, E57 and V63, would provide clues toward elucidating how the CCR5–MIP-1α interaction affects the progress of autoimmune diseases.

  16. Elucidation of the CCR1- and CCR5-binding modes of MIP-1α by application of an NMR spectra reconstruction method to the transferred cross-saturation experiments.

    Science.gov (United States)

    Yoshiura, Chie; Ueda, Takumi; Kofuku, Yutaka; Matsumoto, Masahiko; Okude, Junya; Kondo, Keita; Shiraishi, Yutaro; Shimada, Ichio

    2015-12-01

    C-C chemokine receptor 1 (CCR1) and CCR5 are involved in various inflammation and immune responses, and regulate the progression of the autoimmune diseases differently. However, the number of residues identified at the binding interface was not sufficient to clarify the differences in the CCR1- and CCR5-binding modes to MIP-1α, because the NMR measurement time for CCR1 and CCR5 samples was limited to 24 h, due to their low stability. Here we applied a recently developed NMR spectra reconstruction method, Conservation of experimental data in ANAlysis of FOuRier, to the amide-directed transferred cross-saturation experiments of chemokine receptors, CCR1 and CCR5, embedded in lipid bilayers of the reconstituted high density lipoprotein, and MIP-1α. Our experiments revealed that the residues on the N-loop and β-sheets of MIP-1α are close to both CCR1 and CCR5, and those in the C-terminal helix region are close to CCR5. These results suggest that the genetic influence of the single nucleotide polymorphisms of MIP-1α that accompany substitution of residues in the C-terminal helix region, E57 and V63, would provide clues toward elucidating how the CCR5-MIP-1α interaction affects the progress of autoimmune diseases.

  17. Kinetic evaluation of highly active supported gold catalysts prepared from monolayer-protected clusters: an experimental Michaelis-Menten approach for determining the oxygen binding constant during CO oxidation catalysis.

    Science.gov (United States)

    Long, Cormac G; Gilbertson, John D; Vijayaraghavan, Ganesh; Stevenson, Keith J; Pursell, Christopher J; Chandler, Bert D

    2008-08-06

    Thiol monolayer-protected Au clusters (MPCs) were prepared using dendrimer templates, deposited onto a high-surface-area titania, and then the thiol stabilizers were removed under H2/N2. The resulting Au catalysts were characterized with transmission electron microscopy, X-ray photoelectron spectroscopy, and infrared spectroscopy of adsorbed CO. The Au catalysts prepared via this route displayed minimal particle agglomeration during the deposition and activation steps. Structural data obtained from the physical characterization of the Au catalysts were comparable to features exhibited from a traditionally prepared standard Au catalyst obtained from the World Gold Council (WGC). A differential kinetic study of CO oxidation catalysis by the MPC-prepared Au and the standard WGC catalyst showed that these two catalyst systems have essentially the same reaction order and Arrhenius apparent activation energies (28 kJ/mol). However, the MPC-prepared Au catalyst shows 50% greater activity for CO oxidation. Using a Michaelis-Menten approach, the oxygen binding constants for the two catalyst systems were determined and found to be essentially the same within experimental error. To our knowledge, this kinetic evaluation is the first experimental determination of oxygen binding by supported Au nanoparticle catalysts under working conditions. The values for the oxygen binding equilibrium constant obtained from the Michaelis-Menten treatment (ca. 29-39) are consistent with ultra-high-vacuum measurements on model catalyst systems and support density functional theory calculations for oxygen binding at corner or edge atoms on Au nanoparticles and clusters.

  18. Compact NMR

    Energy Technology Data Exchange (ETDEWEB)

    Bluemich, Bernhard; Haber-Pohlmeier, Sabina; Zia, Wasif [RWTH Aachen Univ. (Germany). Inst. fuer Technische und Makromolekulare Chemie (ITMC)

    2014-06-01

    Nuclear Magnetic Resonance (NMR) spectroscopy is the most popular method for chemists to analyze molecular structures, while Magnetic Resonance Imaging (MRI) is a non-invasive diagnostic tool for medical doctors that provides high-contrast images of biological tissue. In both applications, the sample (or patient) is positioned inside a large, superconducting magnet to magnetize the atomic nuclei. Interrogating radio-frequency pulses result in frequency spectra that provide the chemist with molecular information, the medical doctor with anatomic images, and materials scientist with NMR relaxation parameters. Recent advances in magnet technology have led to a variety of small permanent magnets to allow compact and low-cost instruments. The goal of this book is to provide an introduction to the practical use of compact NMR at a level nearly as basic as the operation of a smart phone.

  19. Proton and iodine-127 nuclear magnetic resonance studies on the binding of iodide by lactoperoxidase

    Energy Technology Data Exchange (ETDEWEB)

    Sakurada, J.; Takahashi, S.; Shimizu, T.; Hatano, M.; Nakamura, S.; Hosoya, T.

    1987-10-06

    Interaction of an iodide ion with lactoperoxidase was studied by the use of /sup 1/H NMR, /sup 127/I NMR, and optical difference spectrum techniques. /sup 1/H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by /sup 127/I NMR, showing no competition with cyanide. Both /sup 1/H NMR and /sup 127/I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pK/sup a/ value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.

  20. Synthesis and NMR spectral properties of spin-labelled mRNA 5' cap analogue: a new tool for biochemical studies of cap binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Stepinski, Janusz [Division of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Wojcik, Jacek [Laboratory of Biological NMR, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawinskiego Street, 02-106 Warsaw (Poland); Sienkiewicz, Andrzej [Biological Physics Group, Institute of Physics, Polish Academy of Sciences, 32/46 Lotnikow Avenue, 02-668 Warsaw (Poland); Niedzwiecka, Anna [Division of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland)

    2007-07-18

    All eukaryotic messenger RNAs (mRNAs) and small nuclear RNAs (snRNAs) comprise a unique chemical structure called a 'cap', i.e. 7-methylguanosine linked by a 5',5' triphosphate bridge to the first transcribed nucleoside. Biophysical studies of interactions between the RNA 5' terminus and proteins that specifically recognize its structure require suitable chemical cap analogues. For the needs of electron spin resonance spectroscopy, a spin-labelled cap analogue, m{sup 7}GTP-TEMPO, P{sup 1}-(7-methylguanosine-5') P{sup 3}-(2,2,6,6-tetramethyl-1-piperidinyloxy-4) triphosphate, has been synthesized and fully characterized spectroscopically. The structure has been confirmed by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR), electron spin resonance (ESR) and electrospray ionization mass spectrometry (ESI-MS). Despite the presence of a free radical (TEMPO) in the small molecule, complete {sup 1}H, {sup 13}C, and {sup 31}P NMR spectra have been acquired that allowed us to assign all these resonances, including the radical moiety. These are the first well resolved NMR spectra of the TEMPO-containing paramagnetic species, directly obtained and analysed without conversion to an N-hydroxylamine derivative.

  1. Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End Binding Protein EB1

    Science.gov (United States)

    Yan, Si; Hou, Guangjin; Schwieters, Charles D.; Ahmed, Shubbir; Williams, John C.; Polenova, Tatyana

    2013-01-01

    Microtubules (MTs) and their associated proteins (MAPs) play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on MAP/MT interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the 3D structure of CAP-Gly domain of mammalian dynactin by MAS NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions. PMID:23648839

  2. A reassessment of the association between azulene and [60]fullerene. Possible pitfalls in the determination of binding constants through fluorescence spectroscopy.

    Science.gov (United States)

    Stella, Lorenzo; Capodilupo, Agostina L; Bietti, Massimo

    2008-10-21

    We show here that the recently reported surprisingly large association constant (K = 7.6 x 10(4) M(-1)) between azulene and [60]fullerene is due to experimental artifacts, pointing out potential errors in the characterization of association equilibria by fluorescence spectroscopy, and suggesting the best experimental practices.

  3. Insight Into Folding,Binding and Stability of Insulin by NMR%胰岛素折叠、结合与稳定性的核磁共振研究

    Institute of Scientific and Technical Information of China (English)

    华庆新

    2004-01-01

    Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and hexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a "native-like B chain super-secondary structure", which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short α-helix at C-terminal of A chain linked. The "super-secondary structure" might be the "folding nucleus" in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C.elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin super-family adopt the "insulin fold" mostly. The structural study of insulin-insulin receptor complex, that of C.elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding

  4. PFG NMR investigations of TPA-TMA-silica mixtures.

    Science.gov (United States)

    Li, Xiang; Shantz, Daniel F

    2011-04-05

    Pulsed-field gradient (PFG) NMR studies of tetrapropylammonium (TPA)-tetramethylammonium (TMA)-silica mixtures are presented, and the effect of TMA as a foreign ion on the TPA-silica nanoparticle interactions before and after heating has been studied. Dynamic light scattering (DLS) results suggest that silica nanoparticles in these TPA-TMA systems grow via a ripening mechanism for the first 24 h of heating. PFG NMR of mixtures before heating show that TMA can effectively displace TPA from the nanoparticle surface. The binding isotherms of TPA at room temperature obtained via PFG NMR can be described by Langmuir isotherms, and indicate a decrease in the adsorbed amount of TPA upon addition of TMA. PFG NMR also shows a systematic increase in the self-diffusion coefficient of TPA in both the mixed TPA-TMA systems and pure TPA systems with heating time, indicating an increased amount of TPA in solution upon heating. By contrast, a much smaller amount of TMA is observed to desorb from the nanoparticles upon heating. These results point to the desorption of TPA from the nanoparticles being a kinetically controlled process. The apparent desorption rate constants were calculated from fitting the desorbed amount of TPA with time via a pseudosecond-order kinetic model. This analysis show the rate of TPA desorption in TPA-TMA mixtures increases with increasing TMA content, whereas for pure TPA mixtures the rate of TPA desorption is much less sensitive to the TPA concentration.

  5. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    Directory of Open Access Journals (Sweden)

    Robert eRoot-Bernstein

    2014-07-01

    Full Text Available Rationale: Insulin resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome and obesity. The mechanism by which insulin and estrogen interact is unknown. We hypothesize that estrogen binds directly to insulin and the insulin receptor producing insulin resistance.Objectives: To determine the binding constants of steroid hormones to insulin, the insulin receptor, and insulin-like peptides derived from the insulin receptor; and to investigate the effect of estrogens on the binding of insulin to its receptor.Methods: Ultraviolet spectroscopy, capillary electrophoresis and NMR demonstrated estrogen binding to insulin and its receptor. Horse-radish peroxidase-linked insulin was used in an ELISA-like procedure to measure the effect of estradiol on binding of insulin to its receptor. Measurements: Binding constants for estrogens to insulin and the insulin receptor were determined by concentration-dependent spectral shifts. The effect of estradiol on insulin-HRP binding to its receptor was determined by shifts in the insulin binding curve. Main Results: Estradiol bound to insulin with a Kd of 12 x 10-9 M and to the insulin receptor with a Kd of 24 x 10-9 M, while other hormones had significantly less affinity. 200 nM estradiol shifted the binding curve of insulin to its receptor 0.8 log units to the right. Conclusions: Estradiol concentrations in many hyperestrogenemic syndromes are sufficient to interfere with insulin binding to its receptor producing significant insulin resistance.

  6. NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

    DEFF Research Database (Denmark)

    Underhaug, Jarl; Jakobsen, Louise Odgaard; Esmann, Mikael;

    2006-01-01

    The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less ......-structured transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport....

  7. NMR Structure of rALF-Pm3, an Anti-Lipopolysaccharide Factor from Shrimp: Model of the Possible Lipid A-Binding Site

    OpenAIRE

    Yang, Yinshan; Boze, Helene; Chemardin, Patrick; Padilla, Andre; Moulin, Guy; Tassanakajon, Anchalee; Pugniere, Martine; Roquet, Francoise; Destoumieux Garzon, Delphine; Gueguen, Yannick; Bachere, Evelyne; Aumelas, Andre

    2009-01-01

    The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and N-15 uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C-34-C-55 disulfide bond was shown to be essential for the structure stab...

  8. MATHEMATICAL CONSTANTS.

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, H.P.; Potter, Elinor

    1971-03-01

    This collection of mathematical data consists of two tables of decimal constants arranged according to size rather than function, a third table of integers from 1 to 1000, giving some of their properties, and a fourth table listing some infinite series arranged according to increasing size of the coefficients of the terms. The decimal values of Tables I and II are given to 20 D.

  9. Hemaka's constant

    CERN Document Server

    Sparavigna, Amelia Carolina

    2012-01-01

    As proposed in a previous paper, the decorations of ancient objects can provide some information on the approximate evaluations of constant {\\pi}, the ratio of circumference to diameter. Here we discuss some disks found in the tomb of Hemaka, the chancellor of a king of the First Dynasty of Egypt, about 3000 BC. The discussion is based on measurements of the dimensionless ratio of lengths.

  10. Integrated computational approach to the analysis of NMR relaxation in proteins: application to ps-ns main chain 15N-1H and global dynamics of the Rho GTPase binding domain of plexin-B1.

    Science.gov (United States)

    Zerbetto, Mirco; Buck, Matthias; Meirovitch, Eva; Polimeno, Antonino

    2011-01-20

    An integrated computational methodology for interpreting NMR spin relaxation in proteins has been developed. It combines a two-body coupled-rotator stochastic model with a hydrodynamics-based approach for protein diffusion, together with molecular dynamics based calculations for the evaluation of the coupling potential of mean force. The method is applied to ¹⁵N relaxation of N-H bonds in the Rho GTPase binding (RBD) domain of plexin-B1, which exhibits intricate internal mobility. Bond vector dynamics are characterized by a rhombic local ordering tensor, S, with principal values S₀² and S₂², and an axial local diffusion tensor, D₂, with principal values D(2,||) and D(2,⊥). For α-helices and β-sheets we find that S₀² ~ -0.5 (strong local ordering), -1.2 computational approach for treating NMR relaxation in proteins by combining stochastic modeling and molecular dynamics. The approach developed provides new insights by its application to a protein that experiences complex dynamics.

  11. NMR GHZ

    CERN Document Server

    Laflamme, R; Zurek, W H; Catasti, P; Mariappan, S V S

    1998-01-01

    We describe the creation of a Greenberger-Horne-Zeilinger (GHZ) state of the form |000>+|111> (three maximally entangled quantum bits) using Nuclear Magnetic Resonance (NMR). We have successfully carried out the experiment using the proton and carbon spins of trichloroethylene, and confirmed the result using state tomography. We have thus extended the space of entangled quantum states explored systematically to three quantum bits, an essential step for quantum computation.

  12. NMR spectroscopy using liquid crystal solvents

    CERN Document Server

    Emsley, JW

    2013-01-01

    NMR Spectroscopy using Liquid Crystal Solvents covers the importance of using a liquid crystal solvent in NMR to derive nuclear dipolar spin-spin coupling constants. This book is composed of ten chapters, and begins with a brief description of the features and benefits of liquid crystal in NMR spectroscopic analysis. The succeeding chapters deal with the mode of operation of nuclear spin Hamiltonian for partially oriented molecules and the analysis of NMR spectra of partially oriented molecules, as well as the determination of rigid molecule structure. These topics are followed by discussions

  13. Structural, stability, dynamic and binding properties of the ALS-causing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations.

    Directory of Open Access Journals (Sweden)

    Shixiong Lua

    Full Text Available T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS. Here using CD, NMR and molecular dynamics (MD simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1 unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2 Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3 T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4 EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5 As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.

  14. Binding of copper(II) ions to the polyproline II helices of PEVK modules of the giant elastic protein titin as revealed by ESI-MS, CD, and NMR.

    Science.gov (United States)

    Ma, Kan; Wang, Kuan

    2003-10-01

    Titin, a family of giant elastic proteins, constitutes an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, the titin PEVK segment acts as a molecular spring to generate elasticity as well as sites of adhesion with parallel thin filaments. Previously, we reported that PEVK consists of tandem repeats of 28 residue modules and that the "polyproline II-coil" motif is the fundamental conformational motif of the PEVK module. In order to characterize the factors that may affect and alter the PPII-coil conformational motifs, we have initiated a systematic study of the interaction with divalent cations (Cu2+, Ca2+, Zn2+, and Ni2+) and a conformational profile of PEVK peptides (a representative 28-mer peptide PR: PEPPKEVVPEKKAPVAPPKKPEVPPVKV and its subfragments PR1: kvPEPPKEVVPE, PR2: VPEKKAPVAPPK, PR3: KPEVPPVKV). UV-Vis absorption difference spectra and CD spectra showed that Cu2+ bound to PR1 with high affinity (20 microM), while its binding to PR2 and PR3 as well as the binding of other cations to all four peptides were of lower affinity (>100 microM). Conformational studies by CD revealed that Cu2+ binding to PR1 resulted in a polyproline II to turn transition up to a 1:2 PR1/Cu2+ ratio and a coil to turn transition at higher Cu2+ concentration. ESI-MS provided the stoichiometry of PEVK peptide-Cu2+ complexes at both low and high ion strength, confirming the specific high affinity binding of Cu2+ to PR1 and PR. Furthermore, NMR and ESI-MS/MS fragmentation analysis elucidated the binding sites of the PEVK peptide-Cu2+ complexes at (-2)KVPE2, 8VPE10, 13APV15, and 22EVP24. A potential application of Cu2+ binding in peptide sequencing by mass spectrometry was also revealed. We conclude that Cu2+ binds and bends PEVK peptides to a beta-turn-like structure at specific sites. The specific targeting of Cu2+ towards PPII is likely to be of significant value in elucidating the roles of PPII in titin elasticity as well as in interactions of

  15. Structural Biology of The sequestration & Transport of Heavy Metal Toxins: NMR Structure Determination of Proteins Containing the CYS-X-Y-Metal Binding Motif

    Energy Technology Data Exchange (ETDEWEB)

    Stanley J. Opella

    2004-03-10

    The support from the Department of Energy enabled us to initiate research on several proteins from the bacterial mercury detoxification system; in particular, we were able to determine the structures of MerP and related metal binding sequences. We have also worked on the membrane transport proteins MerF and MerT.

  16. Solid-state NMR [13C,15N] resonance assignments of the nucleotide-Binding Domain of a bacterial Cyclic Nucleotide-Gated Channel

    NARCIS (Netherlands)

    Cukkemane, A.A.; Nand, D.; Gradmann, S.H.E.; Weingarth, M.H.; Kaupp, U.B.; Baldus, M.

    2012-01-01

    Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-sta

  17. The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

    Directory of Open Access Journals (Sweden)

    van Nuland Nico AJ

    2007-04-01

    Full Text Available Abstract Background SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41 binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3, while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3. Results Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several

  18. Solvent-dependent enthalpic versus entropic anion binding by biaryl substituted quinoline based anion receptors.

    Science.gov (United States)

    Sun, Zhan-Hu; Albrecht, Markus; Raabe, Gerhard; Pan, Fang-Fang; Räuber, Christoph

    2015-01-08

    Anion receptors based on an 8-thiourea substituted quinoline with pentafluorinated (1a) or nonfluorinated (1b) biarylamide groups in the 2-position show similar binding of halide anions with somewhat higher association constants for the more acidic fluorinated derivative. Surprisingly, binding affinities for the halides in the case of the nonfluorinated 1b are similar in nonpolar chloroform or polar DMSO as solvent. Thorough thermodynamic investigations based on NMR van't Hoff analysis show that anion binding in chloroform is mainly enthalpically driven. In DMSO, entropy is the driving force for the binding of the ions with replacement of attached solvent.

  19. The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA: insights into the specific and reversible DNA binding mechanism of dimeric transcription factors.

    Science.gov (United States)

    Sauvé, Simon; Tremblay, Luc; Lavigne, Pierre

    2004-09-17

    Basic region-helix1-loop-helix2-leucine zipper (b/H(1)LH(2)/LZ) transcription factors bind specific DNA sequence in their target gene promoters as dimers. Max, a b/H(1)LH(2)/LZ transcription factor, is the obligate heterodimeric partner of the related b/H(1)LH(2)/LZ proteins of the Myc and Mad families. These heterodimers specifically bind E-box DNA sequence (CACGTG) to activate (e.g. c-Myc/Max) and repress (e.g. Mad1/Max) transcription. Max can also homodimerize and bind E-box sequences in c-Myc target gene promoters. While the X-ray structure of the Max b/H(1)LH(2)/LZ/DNA complex and that of others have been reported, the precise sequence of events leading to the reversible and specific binding of these important transcription factors is still largely unknown. In order to provide insights into the DNA binding mechanism, we have solved the NMR solution structure of a covalently homodimerized version of a Max b/H(1)LH(2)/LZ protein with two stabilizing mutations in the LZ, and characterized its backbone dynamics from (15)N spin-relaxation measurements in the absence of DNA. Apart from minor differences in the pitch of the LZ, possibly resulting from the mutations in the construct, we observe that the packing of the helices in the H(1)LH(2) domain is almost identical to that of the two crystal structures, indicating that no important conformational change in these helices occurs upon DNA binding. Conversely to the crystal structures of the DNA complexes, the first 14 residues of the basic region are found to be mostly unfolded while the loop is observed to be flexible. This indicates that these domains undergo conformational changes upon DNA binding. On the other hand, we find the last four residues of the basic region form a persistent helical turn contiguous to H(1). In addition, we provide evidence of the existence of internal motions in the backbone of H(1) that are of larger amplitude and longer time-scale (nanoseconds) than the ones in the H(2) and LZ domain

  20. 1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator. Evidence for a change of the conformation of the OR3 operator on binding.

    Science.gov (United States)

    Kirpichnikov, M P; Hahn, K D; Buck, F; Rüterjans, H; Chernov, B K; Kurochkin, A V; Skryabin, K G; Bayev, A A

    1984-01-01

    The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein. PMID:6233535

  1. Constant time INEPT CT-HSQC (CTi-CT-HSQC) - A new NMR method to measure accurate one-bond J and RDCs with strong 1H-1H couplings in natural abundance.

    Science.gov (United States)

    Yu, Bingwu; van Ingen, Hugo; Freedberg, Darón I

    2013-03-01

    Strong (1)H-(1)H coupling can significantly reduce the accuracy of (1)J(CH) measured from frequency differences in coupled HSQC spectra. Although accurate (1)J(CH) values can be extracted from spectral simulation, it would be more convenient if the same accurate (1)J(CH) values can be obtained experimentally. Furthermore, simulations reach their limit for residual dipolar coupling (RDC) measurement, as many significant, but immeasurable RDCs are introduced into the spin system when a molecule is weakly aligned, thus it is impossible to have a model spin system that truly represents the real spin system. Here we report a new J modulated method, constant-time INEPT CT-HSQC (CTi-CT-HSQC), to accurately measure one-bond scalar coupling constant and RDCs without strong coupling interference. In this method, changing the spacing between the two 180° pulses during a constant time INEPT period selectively modulates heteronuclear coupling in quantitative J fashion. Since the INEPT delays for measuring one-bond carbon-proton spectra are short compared to (3)J(HH), evolution due to (strong) (1)H-(1)H coupling is marginal. The resulting curve shape is practically independent of (1)H-(1)H coupling and only correlated to the heteronuclear coupling evolution. Consequently, an accurate (1)J(CH) can be measured even in the presence of strong coupling. We tested this method on N-acetyl-glucosamine and mannose whose apparent isotropic (1)J(CH) values are significantly affected by strong coupling with other methods. Agreement to within 0.5Hz or better is found between (1)J(CH) measured by this method and previously published simulation data. We further examined the strong coupling effects on RDC measurements and observed an error up to 100% for one bond RDCs using coupled HSQC in carbohydrates. We demonstrate that RDCs can be obtained with higher accuracy by CTi-CT-HSQC, which compensates the limitation of simulation method.

  2. MD and NMR analyses of choline and TMA binding to duplex DNA: on the origins of aberrant sequence-dependent stability by alkyl cations in aqueous and water-free solvents.

    Science.gov (United States)

    Portella, Guillem; Germann, Markus W; Hud, Nicholas V; Orozco, Modesto

    2014-02-26

    It has been known for decades that alkylammonium ions, such as tetramethyl ammonium (TMA), alter the usual correlation between DNA GC-content and duplex stability. In some cases it is even possible for an AT-rich duplex to be more stable than a GC-rich duplex of the same length. There has been much speculation regarding the origin of this aberration in sequence-dependent DNA duplex stability, but no clear resolution. Using a combination of molecular dynamics simulations and NMR spectroscopy we demonstrate that choline (2-hydroxy-N,N,N-trimethylethanaminium) and TMA are preferentially localized in the minor groove of DNA duplexes at A·T base pairs and these same ions show less pronounced localization in the major groove compared to what has been demonstrated for alkali and alkali earth metal ions. Furthermore, free energy calculations show that single-stranded GC-rich sequences exhibit more favorable solvation by choline than single-stranded AT-rich sequences. The sequence-specific nature of choline and TMA binding provides a rationale for the enhanced stability of AT-rich sequences when alkyl-ammonium ions are used as the counterions of DNA. Our combined theoretical and experimental study provides one of the most detailed pictures to date of cations localized along DNA in the solution state, and provides insights that go beyond understanding alkyl-ammonium ion binding to DNA. In particular, because choline and TMA bind to DNA in a manner that is found to be distinct from that previously reported for Na(+), K(+), Mg(2+), and Ca(2+), our results reveal the important but underappreciated role that most other cations play in sequence-specific duplex stability.

  3. Low-temperature UV-visible and NMR spectroscopic investigations of O(2) binding to ((6)L)Fe(II), a ferrous heme bearing covalently tethered axial pyridine ligands.

    Science.gov (United States)

    Ghiladi, Reza A; Karlin, Kenneth D

    2002-05-06

    In this report, we describe the reversible dioxygen reactivity of ((6)L)Fe(II) (1) [(6)L = partially fluorinated tetraphenylporphyrin with covalently appended TMPA moiety; TMPA = tris(2-pyridylmethyl)amine] using a combination of low-temperature UV-vis and multinuclear ((1)H and (2)H) NMR spectroscopies. Complex 1, or its pyrrole-deuterated analogue ((6)L-d(8))Fe(II) (1-d(8)), exhibits downfield shifted pyrrole resonances (delta 28-60 ppm) in all solvents utilized [CH(2)Cl(2), (CH(3))(2)C(O), CH(3)CN, THF], indicative of a five-coordinate high-spin ferrous heme, even when there is no exogenous axial solvent ligand present (i.e., in methylene chloride). Furthermore, ((6)L)Fe(II) (1) exhibits non-pyrrolic upfield and downfield shifted peaks in CH(2)Cl(2), (CH(3))(2)C(O), and CH(3)CN solvents, which we ascribed to resonances arising from the intra- or intermolecular binding of a TMPA-pyridyl arm to the ferrous heme. Upon exposure to dioxygen at 193 K in methylene chloride, ((6)L)Fe(II) (1) [UV-vis: lambda(max) = 433 (Soret), 529 (sh), 559 nm] reversibly forms a dioxygen adduct [UV-vis: lambda(max) = 422 (Soret), 542 nm], formulated as the six-coordinate low-spin [delta(pyrrole) 9.3 ppm, 193 K] heme-superoxo complex ((6)L)Fe(III)-(O(2)(-)) (2). The coordination of the tethered pyridyl arm to the heme-superoxo complex as axial base ligand is suggested. In coordinating solvents such as THF, reversible oxygenation (193 K) of ((6)L)Fe(II) (1) [UV-vis: lambda(max) = 424 (Soret), 542 nm] also occurs to give a similar adduct ((6)L)Fe(III)-(O(2)(-)) (2) [UV-vis: lambda(max) = 418 (Soret), 537 nm. (2)H NMR: delta(pyrrole) 8.9 ppm, 193 K]. Here, we are unable to distinguish between a bound solvent ligand or tethered pyridyl arm as axial base ligand. In all solvents, the dioxygen adducts decompose (thermally) to the ferric-hydroxy complex ((6)L)Fe(III)-OH (3) [UV-vis: lambda(max) = 412-414 (Soret), 566-575 nm; approximately delta(pyrrole) 120 ppm at 193 K]. This study on the O(2

  4. Tacrine derivatives-acetylcholinesterase interaction: 1H NMR relaxation study.

    Science.gov (United States)

    Delfini, Maurizio; Di Cocco, Maria Enrica; Piccioni, Fabiana; Porcelli, Fernando; Borioni, Anna; Rodomonte, Andrea; Del Giudice, Maria Rosaria

    2007-06-01

    Two acetylcholinesterase (AChE) inhibitors structurally related to Tacrine, 6-methoxytacrine (1a) and 9-heptylamino-6-methoxytacrine (1b), and their interaction with Electrophorus Electricus AChE were investigated. The complete assignment of the 1H and 13C NMR spectra of 1a and 1b was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. This study was undertaken to elucidate the interaction modes between AChE and 1a and 1b in solution, using NMR. The interaction between the two inhibitors and AChE was studied by the analysis of the motional parameters non-selective and selective spin-lattice relaxation times, thereby allowing the motional state of 1a and 1b, both free and bound with AChE, to be defined. The relaxation data pointed out the ligands molecular moiety most involved in the binding with AChE. The relevant ligand/enzyme interaction constants were also evaluated for both compounds and resulted to be 859 and 5412M(-1) for 1a and1b, respectively.

  5. Colorimetry and constant-potential coulometry determinations of transferrin-bound iron, total iron-binding capacity, and total iron in serum containing iron-dextran, with use of sodium dithionite and alumina columns.

    Science.gov (United States)

    Jacobs, J C; Alexander, N M

    1990-10-01

    After the parenteral administration of iron-dextran (imferon), the increased total iron concentrations in serum can be determined by atomic absorption spectroscopy and by colorimetric methods involving sodium dithionite, which reductively dissociates iron from the dextran complex. We report that constant-potential coulometry detects only about 55-70% of dextran-bound iron before dithionite reduction and variable amounts after reaction with the reducing agent. In addition, we have developed a procedure for determining transferrin-bound iron, total iron-binding capacity (TIBC), total iron, and dextran-bound iron with the Kodak Ektachem colorimetric system. In determining total serum iron, the sample is first mixed with sodium dithionite, which rapidly dissociates all dextran-bound iron, but does not remove iron from either transferrin or hemoglobin. After the mixture is applied to an Ektachem slide, transferrin-bound iron is released at pH 4 and is detected together with the iron previously bound to dextran. TIBC is determined by mixing serum with ferric citrate in moderate excess and filtering through a small alumina (Al2O3) column, which binds excess free iron and iron-dextran; the iron in the column eluate represents the TIBC. Transferrin-bound iron is determined by applying diluted serum without added ferric citrate to an alumina column and measuring the iron in the column eluate. Dextran-bound iron is equivalent to the difference between total and transferrin-bound iron. Using this method, we found that transferrin iron-binding sites are saturated in vitro by excess iron-dextran less efficiently than by ferric citrate.

  6. Application of NMR Methods to Identify Detection Reagents for Use in the Development of Robust Nanosensors

    Energy Technology Data Exchange (ETDEWEB)

    Cosman, M; Krishnan, V V; Balhorn, R

    2004-04-29

    Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.

  7. N.M.R. study of organo-phosphorus compounds: non equivalence of methylenic protons in the {alpha} position of an asymmetric phosphorus atom. Application to study of coupling constants J{sub P,H} and J{sub H,H}; R.M.N. de composes organo-phosphores: non equivalence de protons methyleniques en {alpha} d'un phosphore asymetrique. Application a l'etude des constantes de couplage J{sub P,H} et J{sub H,H}

    Energy Technology Data Exchange (ETDEWEB)

    Albrand, J.P. [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

    1969-07-01

    Non-equivalent methylenic protons, with respect to an asymmetric center, have been observed in the n.m.r. spectra of some three- and tetra-coordinated phosphorus compounds. The analysis of these spectra yield the following results: in the studied secondary phosphines, the inversion rate at the phosphorus atom is slow on the n.m.r. time scale; the geminal coupling constant, for a free-rotating methylene group attached to a phosphorus atom, is negative; in phosphines the non equivalence of methylenic protons reveals two {sup 2}J{sub P-C-H} coupling constants which differ by about 5 Hz. This result is in agreement with previous studies on cyclic phosphines. In phosphine oxides, the {sup 2}J{sub P-C-H} values are negative. The {sup 3}J{sub H-P-C-H} coupling constant is positive in both phosphines and phosphine oxides. In phosphines, the non-equivalent methylenic protons exhibit two nearly equal values for this coupling constant. (author) [French] La non-equivalence de protons methyleniques observee dans quelques composes phosphores tricoordines et tetracoordines a apporte les resultats suivants, concernant la stereochimie et les constantes de couplage dans ces composes: dans les phosphines secondaires, la structure pyramidale des liaisons issues du phosphore est fixe a l'echelle de temps de mesure de la R.M.N.; la constante de couplage {sup 2}J{sub H-C-H}, pour un methylene en libre rotation en {alpha} d'un atome de phosphore, est negative; dans les phosphines etudiees, la non-equivalence. observee pour les protons methyleniques s'accompagne d'une difference importante (5 Hz) entre les deux constantes de couplage {sup 2}J{sub P-C-H} determinees par l'analyse; ce resultat est en accord avec la stereospecificite deja observee pour ce couplage dans les phosphines cycliques. Les valeurs observees pour {sup 2}J{sub P-C-H} dans les oxydes de phosphines sont negatives. Les valeurs de la constante de couplage {sup 3}J{sub H-P-C-H}, dans les phosphines

  8. LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

    Science.gov (United States)

    Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco

    2013-10-18

    Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies.

  9. A spectroscopic investigations of anticancer drugs binding to bovine serum albumin

    Science.gov (United States)

    Bakkialakshmi, S.; Chandrakala, D.

    2012-03-01

    The binding of anticancer drugs (i) Uracil (U), (ii) 5-Fluorouracil (5FU) and (iii) 5-Chlorouracil (5ClU), to bovine serum albumin (BSA) at two levels of temperature was studied by the fluorescence of quenching method. UV/Vis, time-resolved fluorescence, Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H NMR) and scanning electron microscope (SEM) analyses were also made. Binding constants (Ka) and binding sites (n) at various levels of temperature were calculated. The obtained binding sites were found to be equal to one for all the three quenchers (U, 5FU and 5ClU) at two different temperature levels. Thermodynamic parameters ΔH, ΔG and ΔS have been calculated and were presented in tables. Change in FTIR absorption intensity shows strong binding of anticancer drugs to BSA. Changes in chemical shifts of NMR and fluorescence lifetimes of the drugs indicate the presence of interaction and binding of BSA to anticancer drugs. 1H NMR spectra and SEM photographs also conform this binding.

  10. Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

    Energy Technology Data Exchange (ETDEWEB)

    Leenheer, J.A.; Brown, G.K.; Cabaniss, S.E. [Geological Survey, Denver, CO (United States); MacCarthy, P. [Colorado School of Mines, Golden, CO (United States). Dept. of Chemistry

    1998-08-15

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca{sup 2+}, Cd{sup 2+}, Cu{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca{sup 2+} ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The metal binding fraction was characterized by quantitative {sup 13}C NMR, {sup 1}H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short-chain aliphatic dibasic acid structures. The Ca{sup 2+} binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.

  11. Solid and solution NMR studies of the complexation of Ag + with the trans isomer of captopril: Biological activities of this high blood pressure drug along with its Ag + complex

    Science.gov (United States)

    Isab, Anvarhusein A.; Wazeer, Mohamed I. M.

    2006-09-01

    Complexation of Ag + with captopril, 1-[(2 S)-3-mercapto-2-methylpropionyl]- L-proline, has been studied by 1H and 13C-NMR spectroscopy. The equilibrium constants for the trans to cis isomers of captopril bound to Ag + were measured by 1H NMR spectroscopy. It is observed that the trans isomer of the drug binds more strongly to Ag + between pH 5 and 8, as shown by the broadening of the trans isomer's resonances in 13C NMR spectra on complexation. A monodentate complexation of the trans captopril with Ag + via the thiol site is proposed based on the solid-state NMR and IR data. A superior antimicrobial activity is exhibited by the Cap-Ag(I) complex compared to captopril ligand itself against Heterotrotropic Plate Counts (HPC), Pseudomonas aeruginosa and Fecal streptococcus bacteria.

  12. NMR of lignins

    Science.gov (United States)

    John Ralph; Larry L. Landucci

    2010-01-01

    This chapter will consider the basic aspects and findings of several forms of NMR spectroscopy, including separate discussions of proton, carbon, heteronuclear, and multidimensional NMR. Enhanced focus will be on 13C NMR, because of its qualitative and quantitative importance, followed by NMR’s contributions to our understanding of lignin...

  13. An in vitro model for the in vivo mobilization of cadmium by chelating agents using 113Cd-NMR spectroscopy.

    Science.gov (United States)

    Beaty, J A; Jones, M M; Wilson, D J; Ma, L

    1992-01-01

    An in vitro method, based on 113Cd-NMR spectroscopy, that provides an alternative to the use of animals for an initial screening of cadmium antagonists is presented. The relative values of the effective stability constants of potential chelating antagonists for cadmium are estimated by using 113Cd-NMR spectroscopy to determine the concentrations of the cadmium species involved in appropriate competitive equilibria. This is accomplished via an examination of the competition between the proposed antagonist and EDTA (ethylenediaminetetraacetic acid) for cadmium-113; previously, EDTA has been shown to be capable of removing cadmium from such in vivo binding sites as metallothionein. The reactions proceed via the stepwise addition of three dithiocarbamate groups to the cadmium accompanied by the concurrent stepwise release of donor groups from the EDTA. The resulting 113Cd-NMR data allow for the determination of the overall stability constant for the complex formed between cadmium and N-methyl-D-glucamine dithiocarbamate, iminodiacetic acid dithiocarbamate, proline dithiocarbamate, sarcosine dithiocarbamate. The use of 113Cd-NMR spectroscopy has the potential for providing direct evidence on the effectiveness of chelate antagonists to compete with endogenous ligands for other toxic metal ions. This technique could prove very useful for other compounds that are not stable enough toward acid and/or base to be examined by standard titrimetric methods.

  14. 1H NMR spectra dataset and solid-state NMR data of cowpea (Vigna unguiculata)

    DEFF Research Database (Denmark)

    Alves Filho, Elenilson G.; Silva, Lorena M. A.; Teofilo, Elizita M.

    2017-01-01

    In this article the NMR data from chemical shifts, coupling constants, and structures of all the characterized compounds were provided, beyond a complementary PCA evaluation for the corresponding manuscript (E.G. Alves Filho, L.M.A. Silva, E.M. Teofilo, F.H. Larsen, E.S. de Brito, 2017) [3......]. In addition, a complementary assessment from solid-state NMR data was provided. For further chemometric analysis, numerical matrices from the raw 1H NMR data were made available in Microsoft Excel workbook format (.xls)....

  15. (1)H NMR spectra dataset and solid-state NMR data of cowpea (Vigna unguiculata).

    Science.gov (United States)

    Alves Filho, Elenilson G; Silva, Lorena M A; Teofilo, Elizita M; Larsen, Flemming H; de Brito, Edy S

    2017-04-01

    In this article the NMR data from chemical shifts, coupling constants, and structures of all the characterized compounds were provided, beyond a complementary PCA evaluation for the corresponding manuscript (E.G. Alves Filho, L.M.A. Silva, E.M. Teofilo, F.H. Larsen, E.S. de Brito, 2017) [3]. In addition, a complementary assessment from solid-state NMR data was provided. For further chemometric analysis, numerical matrices from the raw (1)H NMR data were made available in Microsoft Excel workbook format (.xls).

  16. Discovery of an Interleukin 33 Inhibitor by Molecular Docking Simulation and NMR Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Kwan; Cho, Sang Hyun; Mushtaq, Ameeq UI; Jung, Yong Woo; Byun, Youngjoo; Jeon, Young Ho [Korea University, Sejong (Korea, Republic of); Cho, Haelim [T and J Tech Inc., Seoul (Korea, Republic of)

    2016-02-15

    We identified an inhibitor, which is able to bind to the IL-33 and potentially interfere with ST2 binding by means of docking-based virtual screening and 2D NMR binding analyses. Using this compound, the NMR Chemical shift perturbation (CSP) of IL-33 signals were analyzed and mapped on the structure of IL-33, showing that the identified compound binds at the interface of IL-33 and ST2 binding.

  17. Study of the Halogen Bonding between Pyridine and Perfluoroalkyl Iodide in Solution Phase Using the Combination of FTIR and 19F NMR

    Directory of Open Access Journals (Sweden)

    Briauna Hawthorne

    2013-01-01

    Full Text Available Halogen bonding between pyridine and heptafluoro-2-iodopropane (iso-C3F7I/heptafluoro-1-iodopropane (1-C3F7I was studied using a combination of FTIR and 19F NMR. The ring breathing vibration of pyridine underwent a blue shift upon the formation of halogen bonds with both iso-C3F7I and 1-C3F7I. The magnitudes of the shifts and the equilibrium constants for the halogen-bonded complex formation were found to depend not only on the structure of the halocarbon, but also on the solvent. The halogen bond also affected the Cα-F (C-F bond on the center carbon bending and stretching vibrations in iso-C3F7I. These spectroscopic effects show some solvent dependence, but more importantly, they suggest the possibility of intermolecular halogen bonding among iso-C3F7I molecules. The systems were also examined by 19F NMR in various solvents (cyclohexane, hexane, chloroform, acetone, and acetonitrile. NMR dilution experiments support the existence of the intermolecular self-halogen bonding in both iso-C3F7I and 1-C3F7I. The binding constants for the pyridine/perfluoroalkyl iodide halogen bonding complexes formed in various solvents were obtained through NMR titration experiments. Quantum chemical calculations were used to support the FTIR and 19F NMR observations.

  18. [NMR screening of potential inhibitors of Citrobacter freundii methionine].

    Science.gov (United States)

    Batuev, E A; Lizunov, A Iu; Morozova, E A; Klochkov, V V; Anufrieva, N V; Demidkina, T V; Pol'shakov, V I

    2014-01-01

    Methionine γ-lyase [EC 4.4.1.11] participates in a methionine catabolism at a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. Lack of this enzyme at mammals allows consider it as a perspective target for rational antibacterial drug design. Currently in medical practice there are no the preparations based on an inhibition of methionine γ-lyase activity. We present results of the search of potential inhibitors of the enzyme using the NMR screening techniques based on identification of compounds, which able to bind specifically to their biological target. Study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds, capable to interact with enzyme. Identification of binding was carried out by means of saturation transfer difference (STD) spectroscopy and WaterLOGSY technique. At the final stage the experimental assessment of inhibiting ability of the selected compounds in the reaction of γ-elimination of L-methionine catalyzed by methionine γ-lyase was carried out. Binding constants of two leading compounds were determined using the WaterLOGSY method. The research expands structural group of potential inhibitors of methionine γ-lyase and allows approach to the design of the inhibitors with higher efficacy.

  19. Determination of the molecular complexation constant between alprostadil and alpha-cyclodextrin by conductometry: implications for a freeze-dried formulation.

    Science.gov (United States)

    Sheehy, Philip M; Ramstad, Tore

    2005-10-04

    The binding constant between alprostadil (PGE1) and alpha-cyclodextrin (alpha-CD) was determined at four temperatures using conductance measurements. Alpha-cyclodextrin is an excipient material in Caverject dual chamber syringe (DCS) that was added to enhance stability. The binding constant was used to calculate the amount of PGE1 free upon reconstitution and injection, since only the free drug is clinically active. The conductivity measurement is based on a decrease in specific conductance as alprostadil is titrated with alpha-CD. The change in conductivity was plotted versus free ligand concentration (alpha-CD) to generate a binding curve. As the value of the binding constant proved to be dependent on substrate concentration, it is really a pseudo binding constant. A value of 742+/-60 M(-1) was obtained for a 0.5 mM solution of alprostadil at 27 degrees C and a value of 550+/-52 M(-1) at 37 degrees C. These results compare favorably to values previously obtained by NMR and capillary electrophoresis. Calculation of the fraction PGE1 free upon reconstitution and injection show it to approach the desired outcome of one. Hence, the amount of drug delivered by Caverject DCS is nominally equivalent to that delivered by Caverject S. Po., a predecessor product that contains no alpha-cyclodextrin.

  20. Design of lanthanide fingers: compact lanthanide-binding metalloproteins.

    Science.gov (United States)

    am Ende, Christopher W; Meng, Hai Yun; Ye, Mao; Pandey, Anil K; Zondlo, Neal J

    2010-08-16

    Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide-binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a beta beta alpha structural motif. Lanthanide fingers utilize an Asp(2)Glu(2) metal-coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide-binding peptide incorporated the following key elements: 1) residues with high alpha-helix and beta-sheet propensities in the respective secondary structures; 2) an optimized big box alpha-helix N-cap; 3) a Schellman alpha-helix C-cap motif; and 4) an optional D-Pro-Ser type II' beta-turn in the beta-hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25-residue peptide that was a general lanthanide-binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 microM for binding Er(3+). CD spectra of the peptide-lanthanide complexes are similar to those of zinc fingers and other beta beta alpha proteins. Metal binding involves residues from the N-terminal beta-hairpin and the C terminal alpha-helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D-Pro-Ser type II' beta-turn motif could be replaced by Thr-Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF-hand peptide.

  1. NMR structural studies on antifreeze proteins.

    Science.gov (United States)

    Sönnichsen, F D; Davies, P L; Sykes, B D

    1998-01-01

    Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of approximately 1 degree C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice.

  2. NMR at 900 MHz

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ An important factor in the development of solutionstate NMR has always been th e ability to produce stable and homogeneous magnetic fields. As higher and higher field strengths are reached the pressure is growing on manufacturers to produce NMR systems with greatly improved spectral resolution and signal to noise ratio. The introduction of the Varian 900 MHz INOVA system in August 2000 featuring Oxford Instruments 21.1 T magnet represents the latest pioneering development in NMR technology.

  3. NMR studies of the interaction between inner membrane-associated and periplasmic cytochromes from Geobacter sulfurreducens.

    Science.gov (United States)

    Dantas, Joana M; Brausemann, Anton; Einsle, Oliver; Salgueiro, Carlos A

    2017-06-01

    Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium with notable properties and significance in biotechnological applications. Biochemical studies suggest that the inner membrane-associated diheme cytochrome MacA and the periplasmic triheme cytochrome PpcA from G. sulfurreducens can exchange electrons. In this work, NMR chemical shift perturbation measurements were used to map the interface region and to measure the binding affinity between PpcA and MacA. The results show that MacA binds to PpcA in a cleft defined by hemes I and IV, favoring the contact between PpcA heme IV and the MacA high-potential heme. The dissociation constant values indicate the formation of a low-affinity complex between the proteins, which is consistent with the transient interaction observed in electron transfer complexes. © 2017 Federation of European Biochemical Societies.

  4. The inclusion complex of rosmarinic acid into beta-cyclodextrin: A thermodynamic and structural analysis by NMR and capillary electrophoresis.

    Science.gov (United States)

    Aksamija, Amra; Polidori, Ange; Plasson, Raphaël; Dangles, Olivier; Tomao, Valérie

    2016-10-01

    This work focuses on the characterization of the rosmarinic acid (RA)-β-cyclodextrin (CD) complex in aqueous solution by (1)H NMR (1D- and 2D-ROESY), completed with studies by capillary electrophoresis (CE). From the (1)H NMR data, the stoichiometry of the complex was determined by a Job's plot and the binding constant was estimated from a linear regression (Scott's method). At pH 2.9, the results showed that RA binds CD with a 1:1 stoichiometry and a binding constant Kb of 445 (±53) M(-1) or 465 (±81) M(-1) depending on the CD protons (H-5 or H-3) selected for the evaluation. The Kb value was also calculated from the CD-induced chemical shifts of each RA proton in order to collect information on the structure of the complex. The pH dependence of Kb revealed that the RA carboxylic form displays the highest affinity for CD. An investigation by capillary electrophoresis fully confirmed these results. 2D ROESY analysis provided detailed structural information on the complex and showed a strong correlation between H-3 and H-5 of CD and most RA protons. In conclusion, RA, an efficient phenolic antioxidant from rosemary with a marketing authorization, spontaneously forms a relatively stable inclusion complex with CD in water.

  5. DOSY-NMR and raman investigations on the self-aggregation and cyclodextrin complexation of vanillin.

    Science.gov (United States)

    Ferrazza, Ruggero; Rossi, Barbara; Guella, Graziano

    2014-06-26

    Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a phenolic aldehyde with limited solubility in water; in this work, we investigate its self-aggregation, as well as its complexation equilibria with β-cyclodextrin by using nuclear magnetic resonance (NMR) and vibrational spectroscopy. In particular, diffusion-ordered NMR (DOSY) measurements allowing to detect diffusional changes caused by aggregation/inclusion phenomena lead to a reliable estimate of the equilibrium constants of these processes, while Raman spectroscopy was used to further characterize some structural details of vanillin self-aggregates and inclusion complexes. Although the self-association binding constant of vanillin in water was found to be low (K(a) ∼10), dimeric species are not negligible within the investigated range of concentration (3-65 mM); on the other hand, formation of β-cyclodextrin self-aggregates was not detected by DOSY measurements on aqueous solutions of β-cyclodextrin at different concentrations (2-12 mM). Finally, the binding of vanillin with β-cyclodextrin, as measured by the DOSY technique within a narrow range of concentrations (2-15 mM) by assuming the existence of only the monomeric 1:1 vanillin/β-CD complex, was about an order of magnitude higher (K(c) ∼ 90) than self-aggregation. However, the value of the equilibrium constant for this complexation was found to be significantly affected by the analytical concentrations of the host and guest system, thus indicating that K(c) is an "apparent" equilibrium constant.

  6. Real-time NMR studies of electrochemical double-layer capacitors.

    Science.gov (United States)

    Wang, Hao; Köster, Thomas K-J; Trease, Nicole M; Ségalini, Julie; Taberna, Pierre-Louis; Simon, Patrice; Gogotsi, Yury; Grey, Clare P

    2011-12-07

    (11)B NMR spectroscopy has been used to investigate the sorption of BF(4)(-) anions on a highly porous, high surface area carbon, and different binding sites have been identified. By implementing in situ NMR approaches, the migration of ions between the electrodes of the supercapacitors and changes in the nature of ion binding to the surface have been observed in real time.

  7. NMR logging apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, David O; Turner, Peter

    2014-05-27

    Technologies including NMR logging apparatus and methods are disclosed. Example NMR logging apparatus may include surface instrumentation and one or more downhole probes configured to fit within an earth borehole. The surface instrumentation may comprise a power amplifier, which may be coupled to the downhole probes via one or more transmission lines, and a controller configured to cause the power amplifier to generate a NMR activating pulse or sequence of pulses. Impedance matching means may be configured to match an output impedance of the power amplifier through a transmission line to a load impedance of a downhole probe. Methods may include deploying the various elements of disclosed NMR logging apparatus and using the apparatus to perform NMR measurements.

  8. NMR logging apparatus

    Science.gov (United States)

    Walsh, David O; Turner, Peter

    2014-05-27

    Technologies including NMR logging apparatus and methods are disclosed. Example NMR logging apparatus may include surface instrumentation and one or more downhole probes configured to fit within an earth borehole. The surface instrumentation may comprise a power amplifier, which may be coupled to the downhole probes via one or more transmission lines, and a controller configured to cause the power amplifier to generate a NMR activating pulse or sequence of pulses. Impedance matching means may be configured to match an output impedance of the power amplifier through a transmission line to a load impedance of a downhole probe. Methods may include deploying the various elements of disclosed NMR logging apparatus and using the apparatus to perform NMR measurements.

  9. UC Merced NMR Instrumentation Acquisition

    Science.gov (United States)

    2015-06-18

    UC Merced NMR Instrumentation Acquisition For the UC Merced NMR Instrumentation Acquisition proposal, a new 400 MHz and an upgraded 500 MHz NMR ...UC Merced NMR Instrumentation Acquisition Report Title For the UC Merced NMR Instrumentation Acquisition proposal, a new 400 MHz and an upgraded 500...MHz NMR have been delivered, installed, and incorporated into research and two lab courses. While no results from these instruments have been

  10. Ion exchange equilibrium constants

    CERN Document Server

    Marcus, Y

    2013-01-01

    Ion Exchange Equilibrium Constants focuses on the test-compilation of equilibrium constants for ion exchange reactions. The book first underscores the scope of the compilation, equilibrium constants, symbols used, and arrangement of the table. The manuscript then presents the table of equilibrium constants, including polystyrene sulfonate cation exchanger, polyacrylate cation exchanger, polymethacrylate cation exchanger, polysterene phosphate cation exchanger, and zirconium phosphate cation exchanger. The text highlights zirconium oxide anion exchanger, zeolite type 13Y cation exchanger, and

  11. Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

    Science.gov (United States)

    Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

    2017-03-01

    Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M-1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

  12. Surface-active ionic liquids in micellar catalysis: impact of anion selection on reaction rates in nucleophilic substitutions† †Electronic supplementary information (ESI) available: Formulae for calculating aggregation parameters and fitting of kinetic constants and copies of NMR spectra. See DOI: 10.1039/c6cp00493h Click here for additional data file.

    Science.gov (United States)

    Cognigni, Alice; Gaertner, Peter; Zirbs, Ronald; Peterlik, Herwig; Prochazka, Katharina; Schröder, Christian

    2016-01-01

    A series of surface-active ionic liquids based on the 1-dodecyl-3-methylimidazolium cation and different anions such as halides and alkylsulfates was synthesized. The aggregation behavior of these ionic liquids in water was characterized by surface tension, conductivity measurements and UV-Vis spectroscopy in order to determine the critical micelle concentration (CMC) and to provide aggregation parameters. The determination of surface activity and aggregation properties of amphiphilic ionic liquids was accompanied by SAXS studies on selected surface-active ionic liquids. The application of these surface-active ionic liquids with different anions was tested in nucleophilic substitution reactions for the degradation of organophosphorus compounds. Kinetic studies via UV-Vis spectrophotometry showed a strong acceleration of the reaction in the micellar system compared to pure water. In addition, an influence of the anion was observed, resulting in a correlation between the anion binding to the micelle and the reaction rate constants, indicating that the careful choice of the surface-active ionic liquid can considerably affect the outcome of reactions. PMID:27121134

  13. NMR analysis, protonation equilibria and decomposition kinetics of tolperisone.

    Science.gov (United States)

    Orgován, Gábor; Tihanyi, Károly; Noszál, Béla

    2009-12-05

    The rate constants of spontaneous and hydroxide-catalyzed decomposition and the tautomer-specific protonation constants of tolperisone, a classical muscle relaxant were determined. A solution NMR method without any separation techniques was elaborated to quantitate the progress of decomposition. All the rate and equilibrium constants were determined at four different temperatures and the activation parameters were calculated. The molecular mechanism of decomposition is proposed.

  14. Cosmological Constant, Fine Structure Constant and Beyond

    CERN Document Server

    Wei, Hao; Li, Hong-Yu; Xue, Dong-Ze

    2016-01-01

    In this work, we consider the cosmological constant model $\\Lambda\\propto\\alpha^{-6}$, which is well motivated from three independent approaches. As is well known, the evidence of varying fine structure constant $\\alpha$ was found in 1998. If $\\Lambda\\propto\\alpha^{-6}$ is right, it means that the cosmological constant $\\Lambda$ should be also varying. In this work, we try to develop a suitable framework to model this varying cosmological constant $\\Lambda\\propto\\alpha^{-6}$, in which we view it from an interacting vacuum energy perspective. We propose two types of models to describe the evolutions of $\\Lambda$ and $\\alpha$. Then, we consider the observational constraints on these models, by using the 293 $\\Delta\\alpha/\\alpha$ data from the absorption systems in the spectra of distant quasars, and the data of type Ia supernovae (SNIa), cosmic microwave background (CMB), baryon acoustic oscillation (BAO). We find that the model parameters can be tightly constrained to the narrow ranges of ${\\cal O}(10^{-5})$ t...

  15. Benford distributions in NMR

    CERN Document Server

    Bhole, Gaurav; Mahesh, T S

    2014-01-01

    Benford's Law is an empirical law which predicts the frequency of significant digits in databases corresponding to various phenomena, natural or artificial. Although counter intuitive at the first sight, it predicts a higher occurrence of digit 1, and decreasing occurrences to other larger digits. Here we report the Benford analysis of various NMR databases and draw several interesting inferences. We observe that, in general, NMR signals follow Benford distribution in time-domain as well as in frequency domain. Our survey included NMR signals of various nuclear species in a wide variety of molecules in different phases, namely liquid, liquid-crystalline, and solid. We also studied the dependence of Benford distribution on NMR parameters such as signal to noise ratio, number of scans, pulse angles, and apodization. In this process we also find that, under certain circumstances, the Benford analysis can distinguish a genuine spectrum from a visually identical simulated spectrum. Further we find that chemical-sh...

  16. Binding of carboxylate and trimethylammonium salts to octa-acid and TEMOA deep-cavity cavitands

    Science.gov (United States)

    Sullivan, Matthew R.; Sokkalingam, Punidha; Nguyen, Thong; Donahue, James P.; Gibb, Bruce C.

    2017-01-01

    In participation of the fifth statistical assessment of modeling of proteins and ligands (SAMPL5), the strength of association of six guests ( 3- 8) to two hosts ( 1 and 2) were measured by 1H NMR and ITC. Each host possessed a unique and well-defined binding pocket, whilst the wide array of amphiphilic guests possessed binding moieties that included: a terminal alkyne, nitro-arene, alkyl halide and cyano-arene groups. Solubilizing head groups for the guests included both positively charged trimethylammonium and negatively charged carboxylate functionality. Measured association constants ( K a ) covered five orders of magnitude, ranging from 56 M-1 for guest 6 binding with host 2 up to 7.43 × 106 M-1 for guest 6 binding to host 1.

  17. Rovibrational and temperature effects in theoretical studies of NMR parameters

    DEFF Research Database (Denmark)

    Faber, Rasmus; Kaminsky, Jakub; Sauer, Stephan P. A.

    2016-01-01

    The demand for high precision calculations of NMR shieldings (or their related values, chemical shifts δ) and spin-spin coupling constants facilitating and supporting detailed interpretations of NMR spectra increases hand in hand with the development of computational techniques and hardware...... for molecular equilibrium geometries creates a demand for zero point vibrational and temperature corrections. In this chapter we describe briefly the theory behind rovibrational corrections and review then some important contributions to this field....

  18. Study of molecular interactions with 13C DNP-NMR.

    Science.gov (United States)

    Lerche, Mathilde H; Meier, Sebastian; Jensen, Pernille R; Baumann, Herbert; Petersen, Bent O; Karlsson, Magnus; Duus, Jens Ø; Ardenkjaer-Larsen, Jan H

    2010-03-01

    NMR spectroscopy is an established, versatile technique for the detection of molecular interactions, even when these interactions are weak. Signal enhancement by several orders of magnitude through dynamic nuclear polarization alleviates several practical limitations of NMR-based interaction studies. This enhanced non-equilibrium polarization contributes sensitivity for the detection of molecular interactions in a single NMR transient. We show that direct (13)C NMR ligand binding studies at natural isotopic abundance of (13)C gets feasible in this way. Resultant screens are easy to interpret and can be performed at (13)C concentrations below muM. In addition to such ligand-detected studies of molecular interaction, ligand binding can be assessed and quantified with enzymatic assays that employ hyperpolarized substrates at varying enzyme inhibitor concentrations. The physical labeling of nuclear spins by hyperpolarization thus provides the opportunity to devise fast novel in vitro experiments with low material requirement and without the need for synthetic modifications of target or ligands.

  19. Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

    Energy Technology Data Exchange (ETDEWEB)

    Modi, S.; Behere, D.V.; Mitra, S. (Tata Institute of Fundamental Research, Bombay (India))

    1989-05-30

    The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.

  20. Synthesis, characterization, DNA-binding studies and acetylcholinesterase inhibition activity of new 3-formyl chromone derivatives.

    Science.gov (United States)

    Parveen, Mehtab; Malla, Ali Mohammed; Yaseen, Zahid; Ali, Akhtar; Alam, Mahboob

    2014-01-05

    A series of new substituted 3-formyl chromone derivatives (4-6) were synthesized by one step reaction methodology by knoevenagel condensation, structurally similar to known bisintercalators. The new compounds were characterized by IR, (1)H NMR, (13)C NMR, MS and analytical data. The in vitro DNA binding profile of compounds (4-6) was carried out by absorption, fluorescence and viscosity measurements. It was found that synthesized compounds, especially compound 6 (evident from binding constant value) bind strongly with calf thymus DNA, presumably via an intercalation mode. Additionally, molecular docking studies of compounds (4-6) were carried out with B-DNA (PDBID: 1BNA) which revealed that partial intercalative mode of mechanism is operational in synthesized compounds (4-6) with CT-DNA. The binding constants evaluated from fluorescence spectroscopy of compounds with CT-DNA follows the order compound 6>compound 5>compound 4. All the compounds (4-6) were screened for acetylcholinesterase inhibition assay. It can be inferred from data, that compound (6) showed potent AChE inhibition having IC50=0.27μM, almost in vicinity to reference drug Tacrine (IC50=0.19μM). Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Probing the interaction between cHAVc3 peptide and the EC1 domain of E-cadherin using NMR and molecular dynamics simulations.

    Science.gov (United States)

    Alaofi, Ahmed; Farokhi, Elinaz; Prasasty, Vivitri D; Anbanandam, Asokan; Kuczera, Krzysztof; Siahaan, Teruna J

    2017-01-01

    The goal of this work is to probe the interaction between cyclic cHAVc3 peptide and the EC1 domain of human E-cadherin protein. Cyclic cHAVc3 peptide (cyclo(1,6)Ac-CSHAVC-NH2) binds to the EC1 domain as shown by chemical shift perturbations in the 2D (1)H,-(15)N-HSQC NMR spectrum. The molecular dynamics (MD) simulations of the EC1 domain showed folding of the C-terminal tail region into the main head region of the EC1 domain. For cHAVc3 peptide, replica exchange molecular dynamics (REMD) simulations generated five structural clusters of cHAVc3 peptide. Representative structures of cHAVc3 and the EC1 structure from MD simulations were used in molecular docking experiments with NMR constraints to determine the binding site of the peptide on EC1. The results suggest that cHAVc3 binds to EC1 around residues Y36, S37, I38, I53, F77, S78, H79, and I94. The dissociation constants (Kd values) of cHAVc3 peptide to EC1 were estimated using the NMR chemical shifts data and the estimated Kds are in the range of .5 × 10(-5)-7.0 × 10(-5) M.

  2. Dithieno[3,2-a:2',3'-c]phenazine-based Chemical Probe for Anions: A Spectroscopic Study of Binding

    KAUST Repository

    El-Assaad, Tarek H.

    2015-04-27

    The synthesis of a new anion-responsive molecule N,N\\'-(2,5-bis(4-(tert-butyl)phenyl)dithieno[3,2-a:2\\',3\\'-c]phenazine-9,10-diyl)bis(4-methylbenzenesulfonamide) (1) is reported. The sensitivities of the spectroscopic properties of 1 in the presence of various anions were examined using UV-vis absorption spectroscopy, fluorescence and 1H NMR titration experiments. Strong binding of 1 to carboxylate, cyanide, fluoride and dihydrogen phosphate anions results in an increase in quantum yield for emission of 1, and changes in its 1H NMR chemical shifts. A significant electrostatic interaction of the tetrabutylammonium cation with 1, upon strong binding with the counter anion, was also indicated by the chemical shifts observed in the 1H NMR titrations. Binding constants of 1 to anions are also calculated based on the binding isotherms derived from NMR and UV-Vis titrations. DFT calculations show that the anion does not significantly impact the HOMO/LUMO levels (and subsequently the S0 -> S1 transition), but rather changes the strength of the S0 -> S2 transition, which accounts for the observed changes in the UV-vis spectra.

  3. Generalized Pickands constants

    NARCIS (Netherlands)

    Debicki, K.G.

    2001-01-01

    Pickands constants play an important role in the exact asymptotic of extreme values for Gaussian stochastic processes. By the {it generalized Pickands constant ${cal H_{eta$ we mean the limit begin{eqnarray* {cal H_{eta= lim_{T to inftyfrac{ {cal H_{eta(T){T, end{eqnarray* where ${cal H_{eta(T)= Exp

  4. Synthesis of L-cystine bridged bis(β-cyclodextrin) and its cooperative binding toward guest molecules

    Institute of Scientific and Technical Information of China (English)

    LI Li; LI Xiaoyun; LIU Yu

    2004-01-01

    A novel β-cyclodextrin derivative, L-cystine bridged bis(β-cyclodextrin) 1 has been synthesized and characterized by 1H NMR, 13C NMR, IR, Raman and combustion analyses. Spectrofluorometric titrations have been performed in aqueous phosphate buffer solution (pH=7.20) at 25℃ to give the complex stability constants (Ks) and Gibbs free energy changes (-△G°) for the stoichiometric 1︰1 inclusion complexation of 1 with four dyes. The binding abilities and molecular selectivities are discussed from the viewpoint of size/shape-fit and electrostatic interactions between hosts and guests. 2D ROESY investigation for the complexation between host and guest further confirms the cooperative binding of bridged cyclodextrin 1 with guest.

  5. Teaching NMR Using Online Textbooks

    Directory of Open Access Journals (Sweden)

    Joseph P. Hornak

    1999-12-01

    Full Text Available Nuclear magnetic resonance (NMR spectroscopy has almost become an essential analytical tool for the chemist. High-resolution one- and multi-dimensional NMR, timedomain NMR, and NMR microscopy are but a few of the NMR techniques at a chemist's disposal to determine chemical structure and dynamics. Consequently, even small chemistry departments are finding it necessary to provide students with NMR training and experience in at least some of these techniques. The hands-on experience is readily provided with access to state-of-the-art commercial spectrometers. Instruction in the principles of NMR is more difficult to achieve as most instructors try to teach NMR using single organic or analytical chemistry book chapters with static figures. This paper describes an online textbook on NMR spectroscopy called The Basics of NMR (http://www.cis.rit.edu/htbooks/nmr/ suitable for use in teaching the principles of NMR spectroscopy. The book utilizes hypertext and animations to present the principles of NMR spectroscopy. The book can be used as a textbook associated with a lecture or as a stand-alone teaching tool. Conference participants are encouraged to review the textbook and evaluate its suitability for us in teaching NMR spectroscopy to undergraduate chemistry majors.

  6. The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.

    Directory of Open Access Journals (Sweden)

    Peter M Jones

    Full Text Available ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs, which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.

  7. Nuclear magnetic resonance J coupling constant polarizabilities of hydrogen peroxide

    DEFF Research Database (Denmark)

    Kjær, Hanna; Nielsen, Monia R.; Pagola, Gabriel I.

    2012-01-01

    approximation for the small molecule hydrogen peroxide, which allowed us to carry out calculations with the largest available basis sets optimized for the calculation of NMR coupling constants. We ¿nd a systematic but rather slow convergence with the one-electron basis set and that augmentation functions...

  8. SUMO-1 possesses DNA binding activity

    Directory of Open Access Journals (Sweden)

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  9. Perturbed Angular Correlation (PAC)/NMR spectroscopic properties and dynamics of compounds containing metal ions

    DEFF Research Database (Denmark)

    Arcisauskaité, Vaida

    steps towards understanding how Zn(II) reaches its target position in biological systems in vivo and in vitro experiments in aqueous solution, is the detailed investigation of water exchange reactions for Zn(II)(aq). A very advanced (albeit not complete) picture of structure and dynamics of solvated Zn......199mHg PAC and 199Hg NMR spectroscopic properties, nuclear quadrupole coupling constants, Q, asymmetry parameters, , and chemical shifts, , respectively, are the fingerprint of the local molecular and electronic structure, at the probed Hg nuclei. For this reason, these spectroscopic techniques...... compounds in terms of the atomic constituents. The analysis provided a chemophysical interpretation of changes in Vzz upon structural distortions and ligand exchange. The gained insights can be useful when predicting and understanding changes in Q values for Hg binding sites in proteins. One of the first...

  10. Identification of B. anthracis N(5)-carboxyaminoimidazole ribonucleotide mutase (PurE) active site binding compounds via fragment library screening.

    Science.gov (United States)

    Lei, Hao; Jones, Christopher; Zhu, Tian; Patel, Kavankumar; Wolf, Nina M; Fung, Leslie W-M; Lee, Hyun; Johnson, Michael E

    2016-02-15

    The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme.

  11. NMR, Water and Plants

    NARCIS (Netherlands)

    As, van H.

    1982-01-01

    This Thesis describes the application of a non-destructive pulsed proton NMR method mainly to measure water transport in the xylem vessels of plant stems and in some model systems. The results are equally well applicable to liquid flow in other biological objects than plants, e.g. flow of blood and

  12. Single-sided NMR

    CERN Document Server

    Casanova, Federico; Blümich, Bernhard

    2011-01-01

    Single-Sided NMR describes the design of the first functioning single-sided tomograph, the related measurement methods, and a number of applications. One of the key advantages to this method is the speed at which the images are obtained.

  13. Autonomous driving in NMR.

    Science.gov (United States)

    Perez, Manuel

    2017-01-01

    The automatic analysis of NMR data has been a much-desired endeavour for the last six decades, as it is the case with any other analytical technique. This need for automation has only grown as advances in hardware; pulse sequences and automation have opened new research areas to NMR and increased the throughput of data. Full automatic analysis is a worthy, albeit hard, challenge, but in a world of artificial intelligence, instant communication and big data, it seems that this particular fight is happening with only one technique at a time (let this be NMR, MS, IR, UV or any other), when the reality of most laboratories is that there are several types of analytical instrumentation present. Data aggregation, verification and elucidation by using complementary techniques (e.g. MS and NMR) is a desirable outcome to pursue, although a time-consuming one if performed manually; hence, the use of automation to perform the heavy lifting for users is required to make the approach attractive for scientists. Many of the decisions and workflows that could be implemented under automation will depend on the two-way communication with databases that understand analytical data, because it is desirable not only to query these databases but also to grow them in as much of an automatic manner as possible. How these databases are designed, set up and the data inside classified will determine what workflows can be implemented. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Fully automated system for pulsed NMR measurements

    Energy Technology Data Exchange (ETDEWEB)

    Cantor, David Milton

    1977-01-01

    A system is described which places many of the complex, tedious operations for pulsed NMR experiments under computer control. It automatically optimizes the experiment parameters of pulse length and phase, and precision, accuracy, and measurement speed are improved. The hardware interface between the computer and the NMR instrument is described. Design features, justification of the choices made between alternative design strategies, and details of the implementation of design goals are presented. Software features common to all the available experiments are discussed. Optimization of pulse lengths and phases is performed via a sequential search technique called Uniplex. Measurements of the spin-lattice and spin-spin relaxation times and of diffusion constants are automatic. Options for expansion of the system are explored along with some of the limitations of the system.

  15. Some nitrogen-14 NMR studies in solids

    Energy Technology Data Exchange (ETDEWEB)

    Pratum, T.K.

    1983-11-01

    The first order quadrupolar perturbation of the /sup 14/N NMR spectrum yields information regarding the static and dynamic properties of the surrounding electronic environment. Signal to noise problems caused by long /sup 14/N longitudinal relaxation times (T/sub 1/) and small equilibrium polarizations are reduced by rotating frame cross polarization (CP) experiments between /sup 14/N and /sup 1/H. Using quadrupolar echo and CP techniques, the /sup 14/N quadrupolar coupling constants (e/sup 2/qQ/h) and asymmetry parameters (eta) have been obtained for a variety of tetraalkylammonium compounds by observation of their quadrupolar powder patterns at various temperatures. For choline chloride and iodide the /sup 14/N NMR powder patterns exhibit the effects of anisotropic molecular motion, while choline bromide spectra show no such effects.

  16. NMR and the local structure of relaxors

    Directory of Open Access Journals (Sweden)

    Blinc R.

    2002-01-01

    Full Text Available The relaxor transition in cubic perovskite relaxors (PMN, PSN and PST and tungsten bronze relaxor (SBN has been studied by NMR. The observed spectra are composed of a narrow -1/2 « 1/2 central transition superimposed on a broad background due to satellite transitions. The chemical heterogeneity, responsible for relaxor properties, is reflected here in the structure of the central transition part. The latter is composed of two components, one due to ordered and the other due to disordered regions. Despite of the fact that the macroscopic symmetry does not change when relaxor transition occurs, a non-zero quadruple coupling constant determined from NMR clearly demonstrates the broken local symmetry.

  17. Soils, Pores, and NMR

    Science.gov (United States)

    Pohlmeier, Andreas; Haber-Pohlmeier, Sabina; Haber, Agnes; Sucre, Oscar; Stingaciu, Laura; Stapf, Siegfried; Blümich, Bernhard

    2010-05-01

    Within Cluster A, Partial Project A1, the pore space exploration by means of Nuclear Magnetic Resonance (NMR) plays a central role. NMR is especially convenient since it probes directly the state and dynamics of the substance of interest: water. First, NMR is applied as relaxometry, where the degree of saturation but also the pore geometry controls the NMR signature of natural porous systems. Examples are presented where soil samples from the Selhausen, Merzenhausen (silt loams), and Kaldenkirchen (sandy loam) test sites are investigated by means of Fast Field Cycling Relaxometry at different degrees of saturation. From the change of the relaxation time distributions with decreasing water content and by comparison with conventional water retention curves we conclude that the fraction of immobile water is characterized by T1 samples (Haber-Pohlmeier et al. 2010). Third, relaxometric information forms the basis of understanding magnetic resonance imaging (MRI) results. The general difficulty of imaging in soils are the inherent fast T2 relaxation times due to i) the small pore sizes, ii) presence of paramagnetic ions in the solid matrix, and iii) diffusion in internal gradients. The last point is important, since echo times can not set shorter than about 1ms for imaging purposes. The way out is either the usage of low fields for imaging in soils or special ultra-short pulse sequences, which do not create echoes. In this presentation we will give examples on conventional imaging of macropore fluxes in soil cores (Haber-Pohlmeier et al. 2010), and the combination with relaxometric imaging, as well as the advantages and drawbacks of low-field and ultra-fast pulse imaging. Also first results on the imaging of soil columns measured by SIP in Project A3 are given. Haber-Pohlmeier, S., S. Stapf, et al. (2010). "Waterflow Monitored by Tracer Transport in Natural Porous Media Using MRI." Vadose Zone J.: submitted. Haber-Pohlmeier, S., S. Stapf, et al. (2010). "Relaxation in a

  18. Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

    Science.gov (United States)

    Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

    2016-06-21

    Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

  19. 13C cpmas nmr and molecular modeling in the studies of new analogues of buspirone.

    Science.gov (United States)

    Pisklak, Maciej; Perliński, Mirosław; Kossakowski, Jerzy; Wawer, Iwona

    2002-01-01

    Three derivatives of 1,4 dichloro-dibenzo[e,h]-bicyclo[2.2.3]octane-2,3-dicarboximide were examined by 13C CPMAS NMR. Low energy conformations were found by a semi-empirical AM1 approach, NMR shielding constants were calculated using the GIAO RHF method.

  20. Experimental Determination of pK[subscript a] Values by Use of NMR Chemical Shifts, Revisited

    Science.gov (United States)

    Gift, Alan D.; Stewart, Sarah M.; Bokashanga, Patrick Kwete

    2012-01-01

    This laboratory experiment, using proton NMR spectroscopy to determine the dissociation constant for heterocyclic bases, has been modified from a previously described experiment. A solution of a substituted pyridine is prepared using deuterium oxide (D[subscript 2]O) as the solvent. The pH of the solution is adjusted and proton NMR spectra are…

  1. Variation of Fundamental Constants

    Science.gov (United States)

    Flambaum, V. V.

    2006-11-01

    Theories unifying gravity with other interactions suggest temporal and spatial variation of the fundamental ``constants'' in expanding Universe. The spatial variation can explain a fine tuning of the fundamental constants which allows humans (and any life) to appear. We appeared in the area of the Universe where the values of the fundamental constants are consistent with our existence. We present a review of recent works devoted to the variation of the fine structure constant α, strong interaction and fundamental masses. There are some hints for the variation in quasar absorption spectra. Big Bang nucleosynthesis, and Oklo natural nuclear reactor data. A very promising method to search for the variation of the fundamental constants consists in comparison of different atomic clocks. Huge enhancement of the variation effects happens in transition between accidentally degenerate atomic and molecular energy levels. A new idea is to build a ``nuclear'' clock based on the ultraviolet transition between very low excited state and ground state in Thorium nucleus. This may allow to improve sensitivity to the variation up to 10 orders of magnitude! Huge enhancement of the variation effects is also possible in cold atomic and molecular collisions near Feshbach resonance.

  2. NMR for chemists and biologists

    CERN Document Server

    Carbajo, Rodrigo J

    2013-01-01

    This book offers a concise introduction to the field of nuclear magnetic resonance or NMR. It presents the basic foundations of NMR in a non-mathematical way and provides an overview of both recent and important biological applications of NMR.

  3. NMR magnet technology at MIT

    Energy Technology Data Exchange (ETDEWEB)

    Williams, J.E.C.; Bobrov, E.S.; Iwasa, Y.; Punchard, W.F.B.; Wrenn, J.; Zhukovsky, A. (Massachusetts Inst. of Tech., Cambridge, MA (United States). Francis Bitter National Magnet Lab.)

    1992-01-01

    The design and construction of high field superconducting NMR magnets has much in common with other types of adiabatic superconducting magnets. However, two issues have a particular relevance to NMR magnets. They are field drift and homogeneity. In this paper the control of these factors in the particular context of high field NMR spectrometer magnets is examined.

  4. Photochromism of indolino-benzopyrans studied by NMR and UV-visible spectroscopy

    Directory of Open Access Journals (Sweden)

    S. Delbaere

    2006-01-01

    is described. The structures of photomerocyanines elucidated by NMR spectroscopy and spectrokinetic data (λmax⁡ of colored form, colorability, and rate constant of bleaching obtained by UV-visible spectroscopy are reported.

  5. Bile salt recognition by human liver fatty acid binding protein.

    Science.gov (United States)

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder.

  6. Elastic constants of calcite

    Science.gov (United States)

    Peselnick, L.; Robie, R.A.

    1962-01-01

    The recent measurements of the elastic constants of calcite by Reddy and Subrahmanyam (1960) disagree with the values obtained independently by Voigt (1910) and Bhimasenachar (1945). The present authors, using an ultrasonic pulse technique at 3 Mc and 25??C, determined the elastic constants of calcite using the exact equations governing the wave velocities in the single crystal. The results are C11=13.7, C33=8.11, C44=3.50, C12=4.82, C13=5.68, and C14=-2.00, in units of 1011 dyncm2. Independent checks of several of the elastic constants were made employing other directions and polarizations of the wave velocities. With the exception of C13, these values substantially agree with the data of Voigt and Bhimasenachar. ?? 1962 The American Institute of Physics.

  7. Luminescence, circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin.

    Science.gov (United States)

    Pasricha, Sharda; Sharma, Deepti; Ojha, Himanshu; Gahlot, Pragya; Pathak, Mallika; Basu, Mitra; Chawla, Raman; Singhal, Sugandha; Singh, Anju; Goel, Rajeev; Kukreti, Shrikant; Shukla, Shefali

    2017-05-16

    Chalcones possess various biological properties, for example, antimicrobial, anti-inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using (1) H NMR (13) C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein-drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non-covalent binding interactions in the protein-ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site-specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Biophysical characterization of Ca(2+)-binding of S100A5 and Ca(2+)-induced interaction with RAGE.

    Science.gov (United States)

    Kim, Iktae; Lee, Ko On; Yun, Young-Joo; Jeong, Jea Yeon; Kim, Eun-Hee; Cheong, Haekap; Ryu, Kyoung-Seok; Kim, Nak-Kyoon; Suh, Jeong-Yong

    2017-01-29

    S100A5 is a calcium-binding protein of S100 family, which represents a major ligand to the receptor for advanced glycation end product (RAGE), a pattern recognition receptor engaged in diverse pathological processes. Here we have characterized calcium binding of S100A5 and the complex formation between S100A5 and RAGE using calorimetry and NMR spectroscopy. S100A5 binds to calcium ions in a sequential manner with the equilibrium dissociation constants (KD) of 1.3 μM and 3.5 μM, which corresponds to the calcium-binding at the C-terminal and N-terminal EF-hands. Upon calcium binding, S100A5 interacts with the V domain of RAGE (RAGE-v) to form a heterotrimer (KD ∼5.9 μM) that is distinct among the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGE-v. Distinct binding mode and stoichiometry of RAGE against different S100 family proteins could be important to modulate diverse RAGE signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Algorithm for structure constants

    CERN Document Server

    Paiva, F M

    2011-01-01

    In a $n$-dimensional Lie algebra, random numerical values are assigned by computer to $n(n-1)$ especially selected structure constants. An algorithm is then created, which calculates without ambiguity the remaining constants, obeying the Jacobi conditions. Differently from others, this algorithm is suitable even for poor personal computer. ------------- En $n$-dimensia algebro de Lie, hazardaj numeraj valoroj estas asignitaj per komputilo al $n(n-1)$ speciale elektitaj konstantoj de strukturo. Tiam algoritmo estas kreita, kalkulante senambigue la ceterajn konstantojn, obeante kondicxojn de Jacobi. Malsimile al aliaj algoritmoj, tiu cxi tauxgas ecx por malpotenca komputilo.

  10. Radiographic constant exposure technique

    DEFF Research Database (Denmark)

    Domanus, Joseph Czeslaw

    1985-01-01

    The constant exposure technique has been applied to assess various industrial radiographic systems. Different X-ray films and radiographic papers of two producers were compared. Special attention was given to fast film and paper used with fluorometallic screens. Radiographic image quality...... was tested by the use of ISO wire IQI's and ASTM penetrameters used on Al and Fe test plates. Relative speed and reduction of kilovoltage obtained with the constant exposure technique were calculated. The advantages of fast radiographic systems are pointed out...

  11. The aliquot constant

    CERN Document Server

    Bosma, Wieb

    2009-01-01

    The average value of log s(n)/n taken over the first N even integers is shown to converge to a constant lambda when N tends to infinity; moreover, the value of this constant is approximated and proven to be less than 0. Here s(n) sums the divisors of n less than n. Thus the geometric mean of s(n)/n, the growth factor of the function s, in the long run tends to be less than 1. This could be interpreted as probabilistic evidence that aliquot sequences tend to remain bounded.

  12. Hydrido-ruthenium cluster complexes as models for reactive surface hydrogen species of ruthenium nanoparticles. Solid-state 2H NMR and quantum chemical calculations.

    Science.gov (United States)

    Gutmann, Torsten; Walaszek, Bernadeta; Yeping, Xu; Wächtler, Maria; del Rosal, Iker; Grünberg, Anna; Poteau, Romuald; Axet, Rosa; Lavigne, Guy; Chaudret, Bruno; Limbach, Hans-Heinrich; Buntkowsky, Gerd

    2010-08-25

    The (2)H quadrupolar interaction is a sensitive tool for the characterization of deuterium-metal binding states. In the present study, experimental solid-state (2)H MAS NMR techniques are used in the investigations of two ruthenium clusters, D(4)Ru(4)(CO)(12) (1) and D(2)Ru(6)(CO)(18) (2), which serve as model compounds for typical two-fold, three-fold, and octahedral coordination sites on metal surfaces. By line-shape analysis of the (2)H MAS NMR measurements of sample 1, a quadrupolar coupling constant of 67 +/- 1 kHz, an asymmetry parameter of 0.67 +/- 0.1, and an isotropic chemical shift of -17.4 ppm are obtained. In addition to the neutral complex, sample 2 includes two ionic clusters, identified as anionic [DRu(6)(CO)(18)](-) (2(-)) and cationic [D(3)Ru(6)(CO)(18)](+) (2(+)). By virtue of the very weak quadrupolar interaction (Quantum chemical DFT calculations at different model structures for these ruthenium clusters were arranged in order to help in the interpretation of the experimental results. It is shown that the (2)H nuclear quadrupolar interaction is a sensitive tool for distinguishing the binding state of the deuterons to the transition metal. Combining the data from the polynuclear complexes with the data from mononuclear complexes, a molecular ruler for quadrupolar interactions is created. This ruler now permits the solid-state NMR spectroscopic characterization of deuterium adsorbed on the surfaces of catalytically active metal nanoparticles.

  13. NMR observation of rotory conformers in flexible-chain polymers

    Energy Technology Data Exchange (ETDEWEB)

    Sadykov, R.K.; Makhiyanov, N.; Kurbatov, V.A.; Savel' ev, V.S.; Kirpichnikov, P.A.

    1987-08-01

    The authors conduct a comprehensive line analysis of the NMR spectra of a number of polymers, including cis-1,4-polyisoprenes, cis-1-4-polybutadiene, polyisobutylene, and polyethylene, in a deuterated benzene solvent. Data are given on hyperfine structure and spin-spin coupling constants along with conformational behavior and a negative Overhauser effect observed in the isomers.

  14. Compassion is a constant.

    Science.gov (United States)

    Scott, Tricia

    2015-11-01

    Compassion is a powerful word that describes an intense feeling of commiseration and a desire to help those struck by misfortune. Most people know intuitively how and when to offer compassion to relieve another person's suffering. In health care, compassion is a constant; it cannot be rationed because emergency nurses have limited time or resources to manage increasing demands.

  15. Efficient and cost-effective experimental determination of kinetic constants and data: the success of a Bayesian systematic approach to drug transport, receptor binding, continuous culture and cell transport kinetics.

    Science.gov (United States)

    Murphy, Emma F; Gilmour, Steven G; Crabbe, M James C

    2004-01-02

    Details about the parameters of kinetic systems are crucial for progress in both medical and industrial research, including drug development, clinical diagnosis and biotechnology applications. Such details must be collected by a series of kinetic experiments and investigations. The correct design of the experiment is essential to collecting data suitable for analysis, modelling and deriving the correct information. We have developed a systematic and iterative Bayesian method and sets of rules for the design of enzyme kinetic experiments. Our method selects the optimum design to collect data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. The rules select features of the design such as the substrate range and the number of measurements. We show here that this method can be directly applied to the study of other important kinetic systems, including drug transport, receptor binding, microbial culture and cell transport kinetics. It is possible to reduce the errors in the estimated parameters and, most importantly, increase the efficiency and cost-effectiveness by reducing the necessary amount of experiments and data points measured.

  16. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  17. Biomolecular recognition mechanisms studied by NMR spectroscopy and MD simulations

    NARCIS (Netherlands)

    Hsu, Shang-Te Danny

    2004-01-01

    This thesis describes the use of solution Nuclear Magnetic Resonance (NMR) spectroscopy and Molecular Dynamics (MD) simulations to study the mechanism of biomolecular recognition with two model systems: i) lipid II-binding lantibiotics (lanthionine-containing antibiotics) and ii) the human immunodef

  18. A new method for multi-exponential inversion of NMR relaxation measurements

    Institute of Scientific and Technical Information of China (English)

    WANG; Zhongdong; XIAO; Lizhi; LIU; Tangyan

    2004-01-01

    A new method for multi-exponential inversion to NMR T1 and T2 relaxation time distributions is suggested and tested. Inversion results are compared with MAP-II which is based on SVD algorithm and widely accepted in the industry. Inversed NMR relaxation spectra that have different pre-assigned relaxation times from echo trains with different SNR confirm that the new method with 16 to 64 equally spaced time constants in logarithm scale will ensure the relaxation distribution. Testing results show that the new inversion algorithm is a valuable tool for rock core NMR experimental analysis and NMR logging data process and interpretation.

  19. The role of electrostatic interactions and solvent polarity on the 15N NMR shielding of azines

    Science.gov (United States)

    Modesto-Costa, Lucas; Gester, Rodrigo M.; Manzoni, Vinícius

    2017-10-01

    The nitrogen-15 nuclear magnetic resonance (15N NMR) shielding of azines is very sensitive to the chemical environment. Theoretically, specific interactions are important on the calculation of their spectroscopic properties. However, the choice of the solvent model for the description of NMR shielding constants is still a subject of discussion. In this context, we analyse the role of electrostatic interactions on 15N NMR shielding as function of solvent polarity using the sequential-Quantum Mechanics/Molecular Mechanics approach methodology. Excellent agreement with experimental data of the NMR shielding was obtained without the inclusion of explicit solvent molecules either for polar or non polar solvents.

  20. Conformational dependence of {sup 13}C shielding and coupling constants for methionine methyl groups

    Energy Technology Data Exchange (ETDEWEB)

    Butterfoss, Glenn L. [New York University, Courant Institute of Mathematical Sciences and the Center for Genomics and Systems Biology (United States); DeRose, Eugene F.; Gabel, Scott A.; Perera, Lalith; Krahn, Joseph M.; Mueller, Geoffrey A.; Zheng Xunhai; London, Robert E., E-mail: London@niehs.nih.go [National Institute of Environmental Health Sciences (NIEHS), NIH, Laboratory of Structural Biology (United States)

    2010-09-15

    Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of {sup 3}J{sub CSCC}, {sup 3}J{sub CSCH}, and the isotropic shielding, {sigma}{sub iso}. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-{sup 13}C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, {sup 3}J{sub CSCC}/{sup 3}J{sub COCC} {approx} 0.7. It is further demonstrated that the {sup 3}J{sub CSCH} coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of {delta}{sup 13}C{epsilon}. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large {approx}100{sup o} {chi}{sup 3} values. The utility of the {delta}{sup 13}C{epsilon} as a basis for estimating the gauche/trans ratio for {chi}{sup 3} is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are

  1. Variation of fundamental constants

    CERN Document Server

    Flambaum, V V

    2006-01-01

    We present a review of recent works devoted to the variation of the fine structure constant alpha, strong interaction and fundamental masses. There are some hints for the variation in quasar absorption spectra, Big Bang nucleosynthesis, and Oklo natural nuclear reactor data. A very promising method to search for the variation of the fundamental constants consists in comparison of different atomic clocks. Huge enhancement of the variation effects happens in transition between accidentally degenerate atomic and molecular energy levels. A new idea is to build a ``nuclear'' clock based on the ultraviolet transition between very low excited state and ground state in Thorium nucleus. This may allow to improve sensitivity to the variation up to 10 orders of magnitude! Huge enhancement of the variation effects is also possible in cold atomic and molecular collisions near Feschbach resonance.

  2. Constant-pressure Blowers

    Science.gov (United States)

    Sorensen, E

    1940-01-01

    The conventional axial blowers operate on the high-pressure principle. One drawback of this type of blower is the relatively low pressure head, which one attempts to overcome with axial blowers producing very high pressure at a given circumferential speed. The Schicht constant-pressure blower affords pressure ratios considerably higher than those of axial blowers of conventional design with approximately the same efficiency.

  3. String Scale Cosmological Constant

    OpenAIRE

    Chalmers, Gordon

    2006-01-01

    The cosmological constant is an unexplained until now phenomena of nature that requires an explanation through string effects. The apparent discrepancy between theory and experiment is enourmous and has already been explained several times by the author including mechanisms. In this work the string theory theory of abolished string modes is documented and given perturbatively to all loop orders. The holographic underpinning is also exposed. The matching with the data of the LIGO and D0 experi...

  4. Studies of metal-biomolecule systems in liquids with beta-detected NMR

    CERN Document Server

    Walczak, Michal

    2017-01-01

    My internship took place within a small research team funded via the European Research Council (ERC Starting Grant: Beta-Drop NMR) at ISOLDE. It was devoted to laser spin-polarization and beta-detected NMR techniques and their future applications in chemistry and biology. I was involved in the design and tests of the beta-NMR spectrometer which will be used in the upcoming experiments. In this way I have been exposed to many topics in physics (atomic and nuclear physics), experimental techniques (vacuum technology, lasers, beta detectors, electronics, DAQ software), as well as chemistry and biology (NMR on metal ions, metal ion binding to biomolecules, quantum chemistry calculations).

  5. The Hubble Constant.

    Science.gov (United States)

    Jackson, Neal

    2015-01-01

    I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. There are two broad categories of measurements. The first uses individual astrophysical objects which have some property that allows their intrinsic luminosity or size to be determined, or allows the determination of their distance by geometric means. The second category comprises the use of all-sky cosmic microwave background, or correlations between large samples of galaxies, to determine information about the geometry of the Universe and hence the Hubble constant, typically in a combination with other cosmological parameters. Many, but not all, object-based measurements give H0 values of around 72-74 km s(-1) Mpc(-1), with typical errors of 2-3 km s(-1) Mpc(-1). This is in mild discrepancy with CMB-based measurements, in particular those from the Planck satellite, which give values of 67-68 km s(-1) Mpc(-1) and typical errors of 1-2 km s(-1) Mpc(-1). The size of the remaining systematics indicate that accuracy rather than precision is the remaining problem in a good determination of the Hubble constant. Whether a discrepancy exists, and whether new physics is needed to resolve it, depends on details of the systematics of the object-based methods, and also on the assumptions about other cosmological parameters and which datasets are combined in the case of the all-sky methods.

  6. Universe of constant

    Science.gov (United States)

    Yongquan, Han

    2016-10-01

    The ideal gas state equation is not applicable to ordinary gas, it should be applied to the Electromagnetic ``gas'' that is applied to the radiation, the radiation should be the ultimate state of matter changes or initial state, the universe is filled with radiation. That is, the ideal gas equation of state is suitable for the Singular point and the universe. Maybe someone consider that, there is no vessel can accommodate radiation, it is because the Ordinary container is too small to accommodate, if the radius of your container is the distance that Light through an hour, would you still think it can't accommodates radiation? Modern scientific determinate that the radius of the universe now is about 1027 m, assuming that the universe is a sphere whose volume is approximately: V = 4.19 × 1081 cubic meters, the temperature radiation of the universe (cosmic microwave background radiation temperature of the universe, should be the closest the average temperature of the universe) T = 3.15k, radiation pressure P = 5 × 10-6 N / m 2, according to the law of ideal gas state equation, PV / T = constant = 6 × 1075, the value of this constant is the universe, The singular point should also equal to the constant Author: hanyongquan

  7. Characterization of lithium coordination sites with magic-angle spinning NMR

    Science.gov (United States)

    Haimovich, A.; Goldbourt, A.

    2015-05-01

    Lithium, in the form of lithium carbonate, is one of the most common drugs for bipolar disorder. Lithium is also considered to have an effect on many other cellular processes hence it possesses additional therapeutic as well as side effects. In order to quantitatively characterize the binding mode of lithium, it is required to identify the interacting species and measure their distances from the metal center. Here we use magic-angle spinning (MAS) solid-state NMR to study the binding site of lithium in complex with glycine and water (LiGlyW). Such a compound is a good enzyme mimetic since lithium is four-coordinated to one water molecule and three carboxylic groups. Distance measurements to carbons are performed using a 2D transferred echo double resonance (TEDOR) MAS solid-state NMR experiment, and water binding is probed by heteronuclear high-resolution proton-lithium and proton-carbon correlation (wPMLG-HETCOR) experiments. Both HETCOR experiments separate the main complex from impurities and non-specifically bound lithium species, demonstrating the sensitivity of the method to probe the species in the binding site. Optimizations of the TEDOR pulse scheme in the case of a quadrupolar nucleus with a small quadrupole coupling constant show that it is most efficient when pulses are positioned on the spin-1/2 (carbon-13) nucleus. Since the intensity of the TEDOR signal is not normalized, careful data analysis that considers both intensity and dipolar oscillations has to be performed. Nevertheless we show that accurate distances can be extracted for both carbons of the bound glycine and that these distances are consistent with the X-ray data and with lithium in a tetrahedral environment. The lithium environment in the complex is very similar to the binding site in inositol monophosphatase, an enzyme associated with bipolar disorder and the putative target for lithium therapy. A 2D TEDOR experiment applied to the bacterial SuhB gene product of this enzyme was designed

  8. NMR methodologies for studying mitochondrial bioenergetics.

    Science.gov (United States)

    Alves, Tiago C; Jarak, Ivana; Carvalho, Rui A

    2012-01-01

    Nuclear magnetic resonance (NMR) spectroscopy is a technique with an increasing importance in the study of metabolic diseases. Its initial important role in the determination of chemical structures (1, 2) has been considerably overcome by its potential for the in vivo study of metabolism (3-5). The main characteristic that makes this technique so attractive is its noninvasiveness. Only nuclei capable of transitioning between energy states, in the presence of an intense and constant magnetic field, are studied. This includes abundant nuclei such as proton ((1)H) and phosphorous ((31)P), as well as stable isotopes such as deuterium ((2)H) and carbon 13 ((13)C). This allows a wide range of applications that vary from the determination of water distribution in tissues (as obtained in a magnetic resonance imaging scan) to the calculation of metabolic fluxes under ex vivo and in vivo conditions without the need to use radioactive tracers or tissue biopsies (as in a magnetic resonance spectroscopy (MRS) scan). In this chapter, some technical aspects of the methodology of an NMR/MRS experiment as well as how it can be used to study mitochondrial bioenergetics are overviewed. Advantages and disadvantages of in vivo MRS versus high-resolution NMR using proton high rotation magic angle spinning (HRMAS) of tissue biopsies and tissue extracts are also discussed.

  9. Time domain NMR evaluation of poly(vinyl alcohol) xerogels

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, Elton Jorge da Rocha; Cavalcante, Maxwell de Paula; Tavares, Maria Ines Bruno, E-mail: mibt@ima.ufrj.br [Universidade Federal do Rio de Janeiro (IMA/UFRJ), Rio de Janeiro, RJ (Brazil). Centro de Tecnologia. Instituto de Macromoleculas Professora Eloisa Mano

    2016-05-15

    Poly(vinyl alcohol) (PVA)-based chemically cross-linked xerogels, both neat and loaded with nanoparticulate hydrophilic silica (SiO{sub 2}), were obtained and characterized mainly through time domain NMR experiments (TD-NMR). Fourier-transform infrared (FT-IR) and wide angle X-ray diffraction (WAXD) analyses were employed as secondary methods. TD-NMR, through the interpretation of the spin-lattice relaxation constant values and related information, showed both cross-linking and nanoparticle influences on PVA matrix. SiO{sub 2} does not interact chemically with the PVA chains, but has effect on its molecular mobility, as investigated via TD-NMR. Apparent energy of activation, spin-lattice time constant and size of spin domains in the sample have almost linear dependence with the degree of cross-linking of the PVA and are affected by the addition of SiO{sub 2}. These three parameters were derived from a single set of TD-NMR experiments, which demonstrates the versatility of the technique for characterization of inorganic-organic hybrid xerogels, an important class of materials. (author)

  10. Conformational analysis of small molecules: NMR and quantum mechanics calculations.

    Science.gov (United States)

    Tormena, Cláudio F

    2016-08-01

    This review deals with conformational analysis in small organic molecules, and describes the stereoelectronic interactions responsible for conformational stability. Conformational analysis is usually performed using NMR spectroscopy through measurement of coupling constants at room or low temperature in different solvents to determine the populations of conformers in solution. Quantum mechanical calculations are used to address the interactions responsible for conformer stability. The conformational analysis of a large number of small molecules is described, using coupling constant measurements in different solvents and at low temperature, as well as recent applications of through-space and through-hydrogen bond coupling constants JFH as tools for the conformational analysis of fluorinated molecules. Besides NMR parameters, stereoelectronic interactions such as conjugative, hyperconjugative, steric and intramolecular hydrogen bond interactions involved in conformational preferences are discussed.

  11. Implications of oxidovanadium(IV) binding to actin.

    Science.gov (United States)

    Ramos, Susana; Almeida, Rui M; Moura, José J G; Aureliano, Manuel

    2011-06-01

    Oxidovanadium(IV), a cationic species (VO(2+)) of vanadium(IV), binds to several proteins, including actin. Upon titration with oxidovanadium(IV), approximately 100% quenching of the intrinsic fluorescence of monomeric actin purified from rabbit skeletal muscle (G-actin) was observed, with a V(50) of 131 μM, whereas for the polymerized form of actin (F-actin) 75% of quenching was obtained and a V(50) value of 320 μM. Stern-Volmer plots were used to estimate an oxidovanadium(IV)-actin dissociation constant, with K(d) of 8.2 μM and 64.1 μM VOSO(4), for G-actin and F-actin, respectively. These studies reveal the presence of a high affinity binding site for oxidovanadium(IV) in actin, producing local conformational changes near the tryptophans most accessible to water in the three-dimensional structure of actin. The actin conformational changes, also confirmed by (1)H NMR, are accompanied by changes in G-actin hydrophobic surface, but not in F-actin. The (1)H NMR spectra of G-actin treated with oxidovanadium(IV) clearly indicates changes in the resonances ascribed to methyl group and aliphatic regions as well as to aromatics and peptide-bond amide region. In parallel, it was verified that oxidovanadium(IV) prevents the G-actin polymerization into F-actin. In the 0-200 μM range, VOSO(4) inhibits 40% of the extent of polymerization with an IC(50) of 15.1 μM, whereas 500 μM VOSO(4) totally suppresses actin polymerization. The data strongly suggest that oxidovanadium(IV) binds to actin at specific binding sites preventing actin polymerization. By affecting actin structure and function, oxidovanadium(IV) might be responsible for many cellular effects described for vanadium.

  12. The Hubble Constant

    Directory of Open Access Journals (Sweden)

    Neal Jackson

    2015-09-01

    Full Text Available I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. There are two broad categories of measurements. The first uses individual astrophysical objects which have some property that allows their intrinsic luminosity or size to be determined, or allows the determination of their distance by geometric means. The second category comprises the use of all-sky cosmic microwave background, or correlations between large samples of galaxies, to determine information about the geometry of the Universe and hence the Hubble constant, typically in a combination with other cosmological parameters. Many, but not all, object-based measurements give H_0 values of around 72–74 km s^–1 Mpc^–1, with typical errors of 2–3 km s^–1 Mpc^–1. This is in mild discrepancy with CMB-based measurements, in particular those from the Planck satellite, which give values of 67–68 km s^–1 Mpc^–1 and typical errors of 1–2 km s^–1 Mpc^–1. The size of the remaining systematics indicate that accuracy rather than precision is the remaining problem in a good determination of the Hubble constant. Whether a discrepancy exists, and whether new physics is needed to resolve it, depends on details of the systematics of the object-based methods, and also on the assumptions about other cosmological parameters and which datasets are combined in the case of the all-sky methods.

  13. Transformer-coupled NMR probe

    Science.gov (United States)

    Utsuzawa, Shin; Mandal, Soumyajit; Song, Yi-Qiao

    2012-03-01

    In this study, we propose an NMR probe circuit that uses a transformer with a ferromagnetic core for impedance matching. The ferromagnetic core provides a strong but confined coupling that result in efficient energy transfer between the sample coil and NMR spectrometer, while not disturbing the B1 field generated by the sample coil. We built a transformer-coupled NMR probe and found that it offers comparable performance (loss NQR.

  14. NMR studies on UPt 3

    Science.gov (United States)

    Kitaoka, Y.; Tou, H.; Ishida, K.; Kimura, N.; Ōnuki, Y.; Yamamoto, E.; Haga, Y.; Maezawa, K.

    2000-06-01

    A complete set of the 195Pt Knight-shift (KS) data on the superconducting (SC) state in UPt 3 identified the spin structure of the Cooper pair corresponding to the multiple SC phases. UPt 3 was acclaimed as the first odd-parity superconductor including a non-unitary pairing state characterized by the two-component d vector like db+ idc at low T and low H [H. Tou et al., Phys. Rev. Lett. 77 (1996) 1374; 80 (1998) 3129]. We have shed further light on these novel results through a comparison with the singlet even-parity anisotropic superconductors CeCu 2Si 2 and UPd 2Al 3. In the singlet pairing state, the fractional decrease in KS below T c, δK obs is independent of the crystal direction. We have found that δ χobs=( NAμ B/ Ahf)δ Kobs where Ahf is the hyperfine coupling constant, is in good agreement with spin susceptibilities χγel calculated from an enhanced electronic specific heat γel and χnmr from the quasiparticle Korringa relation T1TKs2=const. This gives direct evidence that the χs of heavy quasiparticles in CeCu 2Si 2 and UPd 2Al 3 is rather isotropic and decreases to zero as T→0 due to the Cooper-pair formation. On the other hand in UPt 3, the δ χobsb, cs along the b- and c-axis in the non-unitary-pairing state (B phase) are two orders of magnitude smaller than χγel and χnmr. These anomalously small values for δ χobsb, cs may suggest either that the spin degree of freedom in the B phase is not perfectly locked to the a-axis or that χs is not enhanced although γel is. The latter is theoretically pointed out by Ikeda and Miyake [J. Phys. Soc. Japan 66 (1997) 3714] to be possible if 5f electrons in the non-Kramerse singlet ground state for 5f 2 are hybridized with conduction electrons. We need further effort towards coherent understanding of a microscopic mechanism leading to the occurrence of the odd-parity superconductivity in UPt 3.

  15. Spaces of constant curvature

    CERN Document Server

    Wolf, Joseph A

    2010-01-01

    This book is the sixth edition of the classic Spaces of Constant Curvature, first published in 1967, with the previous (fifth) edition published in 1984. It illustrates the high degree of interplay between group theory and geometry. The reader will benefit from the very concise treatments of riemannian and pseudo-riemannian manifolds and their curvatures, of the representation theory of finite groups, and of indications of recent progress in discrete subgroups of Lie groups. Part I is a brief introduction to differentiable manifolds, covering spaces, and riemannian and pseudo-riemannian geomet

  16. Conjoined use of EM and NMR in RNA structure refinement.

    Directory of Open Access Journals (Sweden)

    Zhou Gong

    Full Text Available More than 40% of the RNA structures have been determined using nuclear magnetic resonance (NMR technique. NMR mainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characterization of large RNAs can be difficult for NMR alone. Electron microscopy (EM provides global shape information of macromolecules at nanometer resolution, which should be complementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems—U2/U6 small-nuclear RNA, genome-packing motif (Ψ(CD2 from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorporation of a map restraint, either experimental or generated from known PDB structure, greatly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nucleotide in the torsion angle simulated annealing refinement. As increasing number of macromolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures.

  17. Some specific features of the NMR study of fluid flows

    Science.gov (United States)

    Davydov, V. V.

    2016-07-01

    Some specific features of studying fluid flows with a NMR spectrometer are considered. The consideration of these features in the NMR spectrometer design makes it possible to determine the relative concentrations of paramagnetic ions and measure the longitudinal and transverse relaxation times ( T 1 and T 2, respectively) in fluid flows with an error no larger than 0.5%. This approach allows one to completely avoid errors in determining the state of a fluid from measured relaxation constants T 1 and T 2, which is especially urgent when working with medical suspensions and biological solutions. The results of an experimental study of fluid flows are presented.

  18. Magic Angle Spinning NMR Metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jian Z.

    2016-05-31

    Nuclear Magnetic Resonance (NMR) spectroscopy is a non-destructive, quantitative, reproducible, untargeted and unbiased method that requires no or minimal sample preparation, and is one of the leading analytical tools for metabonomics research [1-3]. The easy quantification and the no need of prior knowledge about compounds present in a sample associated with NMR are advantageous over other techniques [1,4]. 1H NMR is especially attractive because protons are present in virtually all metabolites and its NMR sensitivity is high, enabling the simultaneous identification and monitoring of a wide range of low molecular weight metabolites.

  19. Nuclear Magnetic Shielding Constants from Quantum Mechanical/Molecular Mechanical Calculations Using Polarizable Embedding: Role of the Embedding Potential

    DEFF Research Database (Denmark)

    Steinmann, Casper; Olsen, Jógvan Magnus Haugaard; Kongsted, Jacob

    2014-01-01

    We present NMR shielding constants obtained through quantum mechanical/molecular mechanical (QM/MM) embedding calculations. Contrary to previous reports, we show that a relatively small QM region is sufficient, provided that a high-quality embedding potential is used. The calculated averaged NMR...

  20. Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide.

    Science.gov (United States)

    Gonçalves, Elisabete; Kitas, Eric; Seelig, Joachim

    2005-02-22

    Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting

  1. Evaluation of non-covalent interaction between Seryl-Histidine dipeptide and cyclophilin A using NMR and molecular modeling

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Seryl-Histidine dipeptide(Ser-His) has been previously reported to be capable of cleaving DNAs and carboxyl esters,as well as proteins.The protein cleavage mechanism has not been addressed yet.As an initial step of protein cleavage activity,the non-covalent binding affinity of Ser-His for proteins is a crucial prerequisite.In this work,we took cyclophilin A(CyPA) as a substrate protein,and evaluated the non-covalent interaction between CyPA and Ser-His using a combination of NMR spectroscopy and molecular modeling approach.Two independent Ser-His binding sites on CyPA were detected using 15N-1H heteronuclear single-quantum coherence(HSQC) spectra.Each binding site binds one Ser-His molecule.Dissociation constants,Kd1 and Kd2,were estimated to be 2.07 and 6.66 mmol/L,respectively,indicative of the weak non-covalent interaction between Ser-His and CyPA.Based on molecular modeling results,we suggest that both the α-amino and the side chain hydroxyl group of Ser-His are crucial for the non-covalent interaction between Ser-His and CyPA.This work sheds light on the molecular mechanism of Ser-His and its analogues cleaving proteins.

  2. NMR Studies on Li+, Na+ and K+ Complexes of Orthoester Cryptand o-Me2-1.1.1

    Directory of Open Access Journals (Sweden)

    René-Chris Brachvogel

    2015-08-01

    Full Text Available Cryptands, a class of three-dimensional macrobicyclic hosts ideally suited for accommodating small guest ions, have played an important role in the early development of supramolecular chemistry. In contrast to related two-dimensional crown ethers, cryptands have so far only found limited applications, owing in large part to their relatively inefficient multistep synthesis. We have recently described a convenient one-pot, template synthesis of cryptands based on O,O,O-orthoesters acting as bridgeheads. Here we report variable-temperature, 1H-1D EXSY and titration NMR studies on lithium, sodium, and potassium complexes of one such cryptand (o-Me2-1.1.1. Our results indicate that lithium and sodium ions fit into the central cavity of the cryptand, resulting in a comparably high binding affinity and slow exchange with the bulk. The potassium ion binds instead in an exo fashion, resulting in relatively weak binding, associated with fast exchange kinetics. Collectively, these results indicate that orthoester cryptands such as o-Me2-1.1.1 exhibit thermodynamic and kinetic properties in between those typically found for classical crown ethers and cryptands and that future efforts should be directed towards increasing the binding constants.

  3. Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.

    Science.gov (United States)

    Greenwood, Alexander I; Rogals, Monique J; De, Soumya; Lu, Kun Ping; Kovrigin, Evgenii L; Nicholson, Linda K

    2011-09-01

    The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

  4. Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis

    Energy Technology Data Exchange (ETDEWEB)

    Greenwood, Alexander I.; Rogals, Monique J.; De, Soumya [Cornell University, Department of Molecular Biology and Genetics (United States); Lu, Kun Ping [Cancer Biology Program and Biology of Aging Program, Beth Israel Deaconess Medical Center, Harvard Medical School (United States); Kovrigin, Evgenii L. [Marquette University, Chemistry Department (United States); Nicholson, Linda K., E-mail: lkn2@cornell.edu [Cornell University, Department of Molecular Biology and Genetics (United States)

    2011-09-15

    The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, k{sub cat}{sup cis} and apparent Michaelis constants, K{sub M}{sup App}. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific {sup 13}C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide {sup 13}C-{sup 1}H constant time HSQC spectra to determine k{sub cat}{sup cis}, k{sub cat}{sup trans}, K{sub D}{sup cis}, and K{sub D}{sup trans} for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E{center_dot}trans]/[E{center_dot}cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

  5. NMR studies of the R2 repeat and related peptide fragments of the DNA binding domain of c-Myb. New light on the structure and folding of R2.

    Science.gov (United States)

    Ségalas, I.; Desjardins, S.; Oulyadi, H.; Prigent, Y.; Tribouillard, S.; Bernardi, E.; Schoofs, A. R.; Davoust1, D.; Toma, F.

    1999-10-01

    The solution structure of the R2 repeat of the DNA binding domain of the protooncogene c-Myb contains a N-terminal structural motif comprising two antiparallel helices. The motif is stabilized by interactions involving conserved residues. The recognition region in C-terminal position is flexible. This structure differs from that of R2 of another c-Myb protein. La structure en solution de la répétition R2 du domaine de liaison à l'ADN du protooncogène c-Myb possède un motif à deux hélices antiparallèles dans la moitié N-terminale, stabilisé par des interactions entre résidus conservés. La région de reconnaissance à l'ADN en position C-terminale est flexible. Cette structure diffère de celle montrée pour la répétition R2 d'une autre protéine c-Myb.

  6. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    Science.gov (United States)

    Yan, H G; Tsai, M D

    1991-06-04

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. The Hubble Constant

    Directory of Open Access Journals (Sweden)

    Jackson Neal

    2007-09-01

    Full Text Available I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. In the last 20 years, much progress has been made and estimates now range between 60 and 75 km s^-1 Mpc^-1, with most now between 70 and 75 km s^-1 Mpc^-1, a huge improvement over the factor-of-2 uncertainty which used to prevail. Further improvements which gave a generally agreed margin of error of a few percent rather than the current 10% would be vital input to much other interesting cosmology. There are several programmes which are likely to lead us to this point in the next 10 years.

  8. When constants are important

    Energy Technology Data Exchange (ETDEWEB)

    Beiu, V.

    1997-04-01

    In this paper the authors discuss several complexity aspects pertaining to neural networks, commonly known as the curse of dimensionality. The focus will be on: (1) size complexity and depth-size tradeoffs; (2) complexity of learning; and (3) precision and limited interconnectivity. Results have been obtained for each of these problems when dealt with separately, but few things are known as to the links among them. They start by presenting known results and try to establish connections between them. These show that they are facing very difficult problems--exponential growth in either space (i.e. precision and size) and/or time (i.e., learning and depth)--when resorting to neural networks for solving general problems. The paper will present a solution for lowering some constants, by playing on the depth-size tradeoff.

  9. Constant Proportion Portfolio Insurance

    DEFF Research Database (Denmark)

    Jessen, Cathrine

    2014-01-01

    Portfolio insurance, as practiced in 1987, consisted of trading between an underlying stock portfolio and cash, using option theory to place a floor on the value of the position, as if it included a protective put. Constant Proportion Portfolio Insurance (CPPI) is an option-free variation...... on the theme, originally proposed by Fischer Black. In CPPI, a financial institution guarantees a floor value for the “insured” portfolio and adjusts the stock/bond mix to produce a leveraged exposure to the risky assets, which depends on how far the portfolio value is above the floor. Plain-vanilla portfolio...... insurance largely died with the crash of 1987, but CPPI is still going strong. In the frictionless markets of finance theory, the issuer’s strategy to hedge its liability under the contract is clear, but in the real world with transactions costs and stochastic jump risk, the optimal strategy is less obvious...

  10. Decay constants in geochronology

    Institute of Scientific and Technical Information of China (English)

    IgorM.Villa; PaulR.Renne

    2005-01-01

    Geologic time is fundamental to the Earth Sciences, and progress in many disciplines depends critically on our ability to measure time with increasing accuracy and precision. Isotopic geochronology makes use of the decay of radioactive nuclides as a help to quantify the histories of rock, minerals, and other materials. Both accuracy and precision of radioisotopic ages are, at present, limited by those of radioactive decay constants. Modem mass spectrometers can measure isotope ratios with a precision of 10-4 or better. On the other hand, the uncertainties associated with direct half-life determinations are, in most cases, still at the percent level. The present short note briefly summarizes progress and problems that have been encountered during the Working Group's activity.

  11. β-cyclodextrin and caffeine complexes with natural polyphenols from olive and olive oils: NMR, thermodynamic, and molecular modeling studies.

    Science.gov (United States)

    Rescifina, Antonio; Chiacchio, Ugo; Iannazzo, Daniela; Piperno, Anna; Romeo, Giovanni

    2010-11-24

    Complexes of β-cyclodextrin (β-CD) and caffeine (Caf) with biophenols present in olive and olive oil (tyrosol, hydroxytyrosol, homovanillic acid, 3,4-dihydroxyphenylacetic acid, and protocatechuic acid) were investigated by NMR spectroscopy and thermodynamical-molecular dynamic studies to verify the formation of supermolecular aggregates. The obtained results indicated that the investigated biophenols form inclusion complexes with β-CD in a molar ratio of 1:1 in aqueous solution having binding constant values from 10- to 40-fold bigger than those of the corresponding complexes with Caf. Then, β-CD preferentially encloses the biophenol molecule, decreasing its bitter taste and, at the same time, preserving it against chemical and physical decomposition reactions that occur during storage.

  12. Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin

    Directory of Open Access Journals (Sweden)

    Aaron J. Brown

    2017-02-01

    Full Text Available CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG. CXCL7 exists as monomers and dimers, and dimerization (~50 μM and CXCR2 binding (~10 nM constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.

  13. NMR Studies of 3-Acylcamphor

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    NMR studies of some chiral 3-acyclcamphor were conducted.A complete assignment was given to 3-(4-pyridyl)carbonylcamphor by the 2D NMR technology.Assignments were also given to other b -diketones.The results showed that those 3-acylcamphors exist in the enol forms,while 2-benzoyl menthone exists in diketon form.

  14. NMR in pulsed magnetic field

    KAUST Repository

    Abou-Hamad, Edy

    2011-09-01

    Nuclear magnetic resonance (NMR) experiments in pulsed magnetic fields up to 30.4 T focused on 1H and 93Nb nuclei are reported. Here we discuss the advantage and limitation of pulsed field NMR and why this technique is able to become a promising research tool. © 2011 Elsevier Inc. All Rights Reserved.

  15. Structural Biology: Practical NMR Applications

    CERN Document Server

    Teng, Quincy

    2005-01-01

    This textbook begins with an overview of NMR development and applications in biological systems. It describes recent developments in instrument hardware and methodology. Chapters highlight the scope and limitation of NMR methods. While detailed math and quantum mechanics dealing with NMR theory have been addressed in several well-known NMR volumes, chapter two of this volume illustrates the fundamental principles and concepts of NMR spectroscopy in a more descriptive manner. Topics such as instrument setup, data acquisition, and data processing using a variety of offline software are discussed. Chapters further discuss several routine stategies for preparing samples, especially for macromolecules and complexes. The target market for such a volume includes researchers in the field of biochemistry, chemistry, structural biology and biophysics.

  16. Fundamentals of Protein NMR Spectroscopy

    CERN Document Server

    Rule, Gordon S

    2006-01-01

    NMR spectroscopy has proven to be a powerful technique to study the structure and dynamics of biological macromolecules. Fundamentals of Protein NMR Spectroscopy is a comprehensive textbook that guides the reader from a basic understanding of the phenomenological properties of magnetic resonance to the application and interpretation of modern multi-dimensional NMR experiments on 15N/13C-labeled proteins. Beginning with elementary quantum mechanics, a set of practical rules is presented and used to describe many commonly employed multi-dimensional, multi-nuclear NMR pulse sequences. A modular analysis of NMR pulse sequence building blocks also provides a basis for understanding and developing novel pulse programs. This text not only covers topics from chemical shift assignment to protein structure refinement, as well as the analysis of protein dynamics and chemical kinetics, but also provides a practical guide to many aspects of modern spectrometer hardware, sample preparation, experimental set-up, and data pr...

  17. Application of Multi-Exponential Inversion Method to NMR Measurements

    Institute of Scientific and Technical Information of China (English)

    XiaoLizhi; WangZhongdong; LiuTangyan

    2004-01-01

    A new multi-exponential inversion method for NMR relaxation signals is presented and tested, which is based on a solid iteration rebuild technique (SIRT). The T2 spectra inversed by the new method are compared with MAP-Ⅱ results. The T1 and T2 inversion results with different pre-assigned relaxation times and different SNR show that 16 to 64 logarithm equal spaced time constants is better obviously than MAP-Ⅱ. And in particular, it can ensure the relaxation time distribution when the SNR of the measured signal is very low. The new algorithm has been applied in rock core NMR analysis and NMR logging data process and interpretation.

  18. NMR molecular photography

    CERN Document Server

    Khitrin, A K; Fung, B M; Khitrin, Anatoly K.; Ermakov, Vladimir L.

    2002-01-01

    A procedure is described for storing a 2D pattern consisting of 32x32 = 1024 bits in a spin state of a molecular system and then retrieving the stored information as a stack of NMR spectra. The system used is a nematic liquid crystal, the protons of which act as spin clusters with strong intramolecular interactions. The technique used is a programmable multi-frequency irradiation with low amplitude. When it is applied to the liquid crystal, a large number of coherent long-lived 1H response signals can be excited, resulting in a spectrum showing many sharp peaks with controllable frequencies and amplitudes. The spectral resolution is enhanced by using a second weak pulse with a 90 phase shift, so that the 1024 bits of information can be retrieved as a set of well-resolved pseudo-2D spectra reproducing the input pattern.

  19. Negatively charged crown ethers for binding paraquat in water

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A water soluble negatively charged fluorescent 1,4-benzo-1,5-naphtho-36-crown-10-based host has been devised and synthesized.As shown by proton NMR,ESI mass spectrometry and UV-vis spectroscopy,it binds paraquat with a 1:1 stoichiometry and an association constant of 4.50(±0.02) ×103 M-1 in water.Its complexation with paraquat in water was further investigated by fluorescence emission spectroscopy.The results revealed that when paraquat was added to the water solution of the host,the fluorescence emission of the host was quenched by the charge transfer between the crown ether host and paraquat guest.A similar bis-p-phenylene-34-crown-10-based host was synthesized as a contrasting host.Lacking the naphthalene unit,the second host binds paraquat with a 1:1 stoichiometry and a lower association constant,1.04(±0.04) ×103 M-1,in water.This demonstrated that the naphthalene unit could not only act as a strong fluorescence group but also improve the π-πstacking interactions between the host and guest.

  20. A Novel Cobalt(Ⅲ) Mixed-polypyridyl Complex: Synthesis,Characterization and DNA Binding

    Institute of Scientific and Technical Information of China (English)

    CHEN,Hui-Li(陈绘丽); YANG,Pin(杨频)

    2002-01-01

    A novel complex[Co(phen)2HPIP]Cl3[phen=phenanethroline,HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanethroline]has been synthesized and structurally characterized by elemental analysis,UV,IR and 1H NMR spectroscopies. The interaction of the complex with calf thymus DNA(CT DNA)has been studied using absorption and emission spectroscopy, DNA melting techniques and cyclic voltammetry. The compound shows absorption hypochromicity, fluorescence enhancement and DNA melting temperature increment when binding to CT DNA. CV measurement shows a shift in reduction potential and a change in peak current with addition of DNA.These results prove that the compound inserts into DNA base pairs. The shift of peak potential indicates the ion interaction mode between the complex and DNA. The binding constant of the compound to DNA is 4.37×104. The complex also seems to be an efficient photocleavage reagent.

  1. Relaxation dispersion NMR spectroscopy for the study of protein allostery.

    Science.gov (United States)

    Farber, Patrick J; Mittermaier, Anthony

    2015-06-01

    Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery.

  2. Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

    Science.gov (United States)

    Buchholz, Ina; Karg, Beatrice; Dickerhoff, Jonathan; Sievers-Engler, Adrian; Lämmerhofer, Michael; Weisz, Klaus

    2017-03-09

    A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate non-cooperative ligand binding to form 1:1 and 2:1 complex stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. The structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

  3. Chiral diamine-silver(I)-alkene complexes: a quantum chemical and NMR study

    DEFF Research Database (Denmark)

    Kieken, Elsa; Wiest, Olaf; Helquist, Paul

    2005-01-01

    The ability of chiral diamine silver complexes to bind chiral and prochiral alkenes has been analyzed in detail. The stereoselectivity in binding of alkenes to a chiral ethanediamine silver complex has been investigated by NMR. The low-energy conformations of several small model complexes have be...

  4. 1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

    Science.gov (United States)

    Liu, S; Gresser, M J; Tracey, A S

    1992-03-17

    The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. The Interacting and Non-constant Cosmological Constant

    CERN Document Server

    Verma, Murli Manohar

    2009-01-01

    We propose a time-varying cosmological constant with a fixed equation of state, which evolves mainly through its interaction with the background during most of the long history of the universe. However, such interaction does not exist in the very early and the late-time universe and produces the acceleration during these eras when it becomes very nearly a constant. It is found that after the initial inflationary phase, the cosmological constant, that we call as lambda parameter, rolls down from a large constant value to another but very small constant value and further dominates the present epoch showing up in form of the dark energy driving the acceleration.

  6. Quantum Theory without Planck's Constant

    CERN Document Server

    Ralston, John P

    2012-01-01

    Planck's constant was introduced as a fundamental scale in the early history of quantum mechanics. We find a modern approach where Planck's constant is absent: it is unobservable except as a constant of human convention. Despite long reference to experiment, review shows that Planck's constant cannot be obtained from the data of Ryberg, Davisson and Germer, Compton, or that used by Planck himself. In the new approach Planck's constant is tied to macroscopic conventions of Newtonian origin, which are dispensable. The precision of other fundamental constants is substantially improved by eliminating Planck's constant. The electron mass is determined about 67 times more precisely, and the unit of electric charge determined 139 times more precisely. Improvement in the experimental value of the fine structure constant allows new types of experiment to be compared towards finding "new physics." The long-standing goal of eliminating reliance on the artifact known as the International Prototype Kilogram can be accompl...

  7. Weak intermolecular interactions in gas-phase NMR

    CERN Document Server

    Garbacz, Piotr; Jackowski, Karol; Moszynski, Robert; Jaszunski, Michal

    2011-01-01

    Gas-phase NMR spectra demonstrating the effect of weak intermolecular forces on the NMR shielding constants of the interacting species are reported. We analyse the interaction of the molecular hydrogen isotopomers with He, Ne, and Ar, and the interaction in the He-CO_2 dimer. The same effects are studied for all these systems in the ab initio calculations. The comparison of the experimental and computed shielding constants is shown to depend strongly on the treatment of the bulk susceptibility effects, which determine in practice the pressure dependence of the experimental values. Best agreement of the results is obtained when the bulk susceptibility correction in rare gas solvents is evaluated from the analysis of the He-rare gas interactions, and when the shielding of deuterium in D_2-rare gas systems is considered.

  8. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  9. nmr spectroscopic study and dft calculations of giao nmr shieldings ...

    African Journals Online (AJOL)

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    various fields of science and industry such as microelectronic and aerospace ... GIAO/DFT (Gauge Including Atomic Orbitals/Density Functional Theory) approach is .... successfully by using NMR and quantum chemical calculations.

  10. NMR spectrometers as "magnetic tongues"

    DEFF Research Database (Denmark)

    Malmendal, Anders; Amoresano, Claudia; Trotta, Roberta

    2011-01-01

    opened up the possibility to calibrate the sensory perception. In this frame, we have tested the potentiality of nuclear magnetic resonance spectroscopy as a predictive tool to measure sensory descriptors. In particular, we have used an NMR metabolomic approach that allowed us to differentiate...... the analyzed samples based on their chemical composition. We were able to correlate the NMR metabolomic fingerprints recorded for canned tomato samples to the sensory descriptors bitterness, sweetness, sourness, saltiness, tomato and metal taste, redness, and density, suggesting that NMR might be a very useful...

  11. Annual reports on NMR spectroscopy

    CERN Document Server

    Webb, Graham A; McCarthy, M J

    1995-01-01

    Over recent years, no other technique has grown to such importance as that of NMR spectroscopy. It is used in all branches of science where precise structural determination is required and where the nature of interactions and reactions in solution is being studied. Annual Reports on NMR Spectroscopy has established itself as a means for the specialist and non-specialist alike to become familiar with new applications of the technique in all branches of chemistry, including biochemistry, and pharmaceutics. This volume focuses on theoretical aspects of NMR nuclear shielding and on applications of

  12. Effective rotational correlation times of proteins from NMR relaxation interference

    Science.gov (United States)

    Lee, Donghan; Hilty, Christian; Wider, Gerhard; Wüthrich, Kurt

    2006-01-01

    Knowledge of the effective rotational correlation times, τc, for the modulation of anisotropic spin-spin interactions in macromolecules subject to Brownian motion in solution is of key interest for the practice of NMR spectroscopy in structural biology. The value of τc enables an estimate of the NMR spin relaxation rates, and indicates possible aggregation of the macromolecular species. This paper reports a novel NMR pulse scheme, [ 15N, 1H]-TRACT, which is based on transverse relaxation-optimized spectroscopy and permits to determine τc for 15N- 1H bonds without interference from dipole-dipole coupling of the amide proton with remote protons. [ 15N, 1H]-TRACT is highly efficient since only a series of one-dimensional NMR spectra need to be recorded. Its use is suggested for a quick estimate of the rotational correlation time, to monitor sample quality and to determine optimal parameters for complex multidimensional NMR experiments. Practical applications are illustrated with the 110 kDa 7,8-dihydroneopterin aldolase from Staphylococcus aureus, the uniformly 15N-labeled Escherichia coli outer membrane protein X (OmpX) in 60 kDa mixed OmpX/DHPC micelles with approximately 90 molecules of unlabeled 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DHPC), and the 16 kDa pheromone-binding protein from Bombyx mori, which cover a wide range of correlation times.

  13. NMR study of small molecule adsorption in MOF-74-Mg.

    Science.gov (United States)

    Lopez, M G; Canepa, Pieremanuele; Thonhauser, T

    2013-04-21

    We calculate the carbon nuclear magnetic resonance (NMR) shielding for CO2 and the hydrogen shieldings for both H2 and H2O inside the metal organic framework MOF-74-Mg. Our ab initio calculations are at the density functional theory level using the van der Waals including density functional vdW-DF. The shieldings are obtained while placing the small molecules throughout the structure, including the calculated adsorption site for various loading scenarios. We then explore relationships between loading, rotational and positional characteristics, and the NMR shieldings for each adsorbate. Our NMR calculations show a change in the shielding depending on adsorbate, position, and loading in a range that is experimentally observable. We further provide a simple model for the energy and the NMR shieldings throughout the cavity of the MOF. By providing this mapping of shielding to position and loading for these adsorbates, we argue that NMR probes could be used to provide additional information about the position at which these small molecules bind within the MOF, as well as the loading of the adsorbed molecule.

  14. NMR study of small molecule adsorption in MOF-74-Mg

    Science.gov (United States)

    Lopez, M. G.; Canepa, Pieremanuele; Thonhauser, T.

    2013-04-01

    We calculate the carbon nuclear magnetic resonance (NMR) shielding for CO2 and the hydrogen shieldings for both H2 and H2O inside the metal organic framework MOF-74-Mg. Our ab initio calculations are at the density functional theory level using the van der Waals including density functional vdW-DF. The shieldings are obtained while placing the small molecules throughout the structure, including the calculated adsorption site for various loading scenarios. We then explore relationships between loading, rotational and positional characteristics, and the NMR shieldings for each adsorbate. Our NMR calculations show a change in the shielding depending on adsorbate, position, and loading in a range that is experimentally observable. We further provide a simple model for the energy and the NMR shieldings throughout the cavity of the MOF. By providing this mapping of shielding to position and loading for these adsorbates, we argue that NMR probes could be used to provide additional information about the position at which these small molecules bind within the MOF, as well as the loading of the adsorbed molecule.

  15. Sensitization of a stray-field NMR to vibrations: a potential for MR elastometry with a portable NMR sensor.

    Science.gov (United States)

    Mastikhin, Igor; Barnhill, Marie

    2014-11-01

    An NMR signal from a sample in a constant stray field of a portable NMR sensor is sensitized to vibrations. The CPMG sequence is synchronized to vibrations so that the constant gradient becomes an "effective" square-wave gradient, leading to the vibration-induced phase accumulation. The integrating nature of the spot measurement, combined with the phase distribution due to a non-uniform gradient and/or a wave field, leads to a destructive interference, the drop in the signal intensity and changes in the echo train shape. Vibrations with amplitudes as small as 140 nm were reliably detected with the permanent gradient of 12.4 T/m. The signal intensity depends on the phase offset between the vibrations and the pulse sequence. This approach opens the way for performing elastometry and micro-rheology measurements with portable NMR devices beyond the walls of a laboratory. Even without synchronization, if a vibration frequency is comparable to 1/2TE of the CPMG sequence, the signal can be severely affected, making it important for potential industrial applications of stray-field NMR.

  16. Programming A Molecular Relay for Ultrasensitive Biodetection through 129 Xe NMR

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yanfei [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Roose, Benjamin W. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Philbin, John P. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Doman, Jordan L. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Dmochowski, Ivan J. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA

    2015-12-21

    We reported a supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized 129Xe NMR. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. Moreover, the protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn2+ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive 129Xe NMR spectroscopy.

  17. Programming A Molecular Relay for Ultrasensitive Biodetection through (129)Xe NMR.

    Science.gov (United States)

    Wang, Yanfei; Roose, Benjamin W; Philbin, John P; Doman, Jordan L; Dmochowski, Ivan J

    2016-01-26

    A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized (129) Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]-Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn(2+) in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive (129) Xe NMR spectroscopy.

  18. Integrative NMR for biomolecular research.

    Science.gov (United States)

    Lee, Woonghee; Cornilescu, Gabriel; Dashti, Hesam; Eghbalnia, Hamid R; Tonelli, Marco; Westler, William M; Butcher, Samuel E; Henzler-Wildman, Katherine A; Markley, John L

    2016-04-01

    NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page ( http://pine.nmrfam.wisc.edu/download_packages.html ), and detailed instructions are available from the Integrative NMR Video Tutorial page ( http://pine.nmrfam.wisc.edu/integrative.html ).

  19. Compact orthogonal NMR field sensor

    Science.gov (United States)

    Gerald, II, Rex E.; Rathke, Jerome W [Homer Glen, IL

    2009-02-03

    A Compact Orthogonal Field Sensor for emitting two orthogonal electro-magnetic fields in a common space. More particularly, a replacement inductor for existing NMR (Nuclear Magnetic Resonance) sensors to allow for NMR imaging. The Compact Orthogonal Field Sensor has a conductive coil and a central conductor electrically connected in series. The central conductor is at least partially surrounded by the coil. The coil and central conductor are electrically or electro-magnetically connected to a device having a means for producing or inducing a current through the coil and central conductor. The Compact Orthogonal Field Sensor can be used in NMR imaging applications to determine the position and the associated NMR spectrum of a sample within the electro-magnetic field of the central conductor.

  20. Optical pumping and xenon NMR

    Energy Technology Data Exchange (ETDEWEB)

    Raftery, M. Daniel [Univ. of California, Berkeley, CA (United States)

    1991-11-01

    Nuclear Magnetic Resonance (NMR) spectroscopy of xenon has become an important tool for investigating a wide variety of materials, especially those with high surface area. The sensitivity of its chemical shift to environment, and its chemical inertness and adsorption properties make xenon a particularly useful NMR probe. This work discusses the application of optical pumping to enhance the sensitivity of xenon NMR experiments, thereby allowing them to be used in the study of systems with lower surface area. A novel method of optically-pumping 129Xe in low magnetic field below an NMR spectrometer and subsequent transfer of the gas to high magnetic field is described. NMR studies of the highly polarized gas adsorbed onto powdered samples with low to moderate surface areas are now possible. For instance, NMR studies of optically-pumped xenon adsorbed onto polyacrylic acid show that xenon has a large interaction with the surface. By modeling the low temperature data in terms of a sticking probability and the gas phase xenon-xenon interaction, the diffusion coefficient for xenon at the surface of the polymer is determined. The sensitivity enhancement afforded by optical pumping also allows the NMR observation of xenon thin films frozen onto the inner surfaces of different sample cells. The geometry of the thin films results in interesting line shapes that are due to the bulk magnetic susceptibility of xenon. Experiments are also described that combine optical pumping with optical detection for high sensitivity in low magnetic field to observe the quadrupoler evolution of 131 Xe spins at the surface of the pumping cells. In cells with macroscopic asymmetry, a residual quadrupolar interaction causes a splitting in the 131Xe NMR frequencies in bare Pyrex glass cells and cells with added hydrogen.

  1. Optical pumping and xenon NMR

    Energy Technology Data Exchange (ETDEWEB)

    Raftery, M.D.

    1991-11-01

    Nuclear Magnetic Resonance (NMR) spectroscopy of xenon has become an important tool for investigating a wide variety of materials, especially those with high surface area. The sensitivity of its chemical shift to environment, and its chemical inertness and adsorption properties make xenon a particularly useful NMR probe. This work discusses the application of optical pumping to enhance the sensitivity of xenon NMR experiments, thereby allowing them to be used in the study of systems with lower surface area. A novel method of optically-pumping [sup 129]Xe in low magnetic field below an NMR spectrometer and subsequent transfer of the gas to high magnetic field is described. NMR studies of the highly polarized gas adsorbed onto powdered samples with low to moderate surface areas are now possible. For instance, NMR studies of optically-pumped xenon adsorbed onto polyacrylic acid show that xenon has a large interaction with the surface. By modeling the low temperature data in terms of a sticking probability and the gas phase xenon-xenon interaction, the diffusion coefficient for xenon at the surface of the polymer is determined. The sensitivity enhancement afforded by optical pumping also allows the NMR observation of xenon thin films frozen onto the inner surfaces of different sample cells. The geometry of the thin films results in interesting line shapes that are due to the bulk magnetic susceptibility of xenon. Experiments are also described that combine optical pumping with optical detection for high sensitivity in low magnetic field to observe the quadrupoler evolution of 131 Xe spins at the surface of the pumping cells. In cells with macroscopic asymmetry, a residual quadrupolar interaction causes a splitting in the [sup 131]Xe NMR frequencies in bare Pyrex glass cells and cells with added hydrogen.

  2. NMR characterization of thin films

    Science.gov (United States)

    Gerald, II, Rex E.; Klingler, Robert J.; Rathke, Jerome W.; Diaz, Rocio; Vukovic, Lela

    2008-11-25

    A method, apparatus, and system for characterizing thin film materials. The method, apparatus, and system includes a container for receiving a starting material, applying a gravitational force, a magnetic force, and an electric force or combinations thereof to at least the starting material, forming a thin film material, sensing an NMR signal from the thin film material and analyzing the NMR signal to characterize the thin film of material.

  3. Report on neptunium speciation by NMR and optical spectroscopies

    Energy Technology Data Exchange (ETDEWEB)

    Tait, C.D.; Palmer, P.D.; Ekberg, S.A.; Clark, D.L.

    1995-11-01

    Hydrolysis and carbonate complexation reactions were examined for NpO{sub 2}{sup 2+} and NpO{sub 2}{sup +} ions by a variety of techniques including potentiometric titration, UV-Vis-NIR and NMR spectroscopy. The equilibrium constant for the reaction 3NpO{sub 2}(CO{sub 3}){sub 3}{sup 4{minus}} + 3H{sup +} {rightleftharpoons} (NpO{sub 2}){sub 3}(CO{sub 3}){sub 6}{sup 6{minus}} + 3HCO{sub 3}{sup {minus}} was determined to be logK = 19.7 ({plus_minus} 0.8) (I = 2.5 m). {sup 17}O NMR spectroscopy of NpO{sub 2}{sup n+} ions (n = 1,2) reveals a readily observable {sup 17}O resonance for n = 2, but not for n = 1. The first hydrolysis constant for NpO{sub 2}{sup +} was studied as a function of temperature, and the functional form for the temperature-dependent equilibrium constant for the reaction written as NpO{sub 2}{sup +} + H{sub 2}O {rightleftharpoons} NpO{sub 2}OH + H{sup +} was found to be logK = 2.28 {minus} 3780/T, where T is in {degree}K. Finally, the temperature dependence of neptunium(V) carbonate complexation constants was studied. For the first carbonate complexation constant, the appropriate functional form was found to be log{beta}{sub 01} = 1.47 + 786/T.

  4. Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement.

    Science.gov (United States)

    Calhoun, Jennifer R; Liu, Weixia; Spiegel, Katrin; Dal Peraro, Matteo; Klein, Michael L; Valentine, Kathleen G; Wand, A Joshua; DeGrado, William F

    2008-02-01

    We report the solution NMR structure of a designed dimetal-binding protein, di-Zn(II) DFsc, along with a secondary refinement step employing molecular dynamics techniques. Calculation of the initial NMR structural ensemble by standard methods led to distortions in the metal-ligand geometries at the active site. Unrestrained molecular dynamics using a nonbonded force field for the metal shell, followed by quantum mechanical/molecular mechanical dynamics of DFsc, were used to relax local frustrations at the dimetal site that were apparent in the initial NMR structure and provide a more realistic description of the structure. The MD model is consistent with NMR restraints, and in good agreement with the structural and functional properties expected for DF proteins. This work demonstrates that NMR structures of metalloproteins can be further refined using classical and first-principles molecular dynamics methods in the presence of explicit solvent to provide otherwise unavailable insight into the geometry of the metal center.

  5. Benzocaine complexation with p-sulfonic acid calix[n]arene: experimental ((1) H-NMR) and theoretical approaches.

    Science.gov (United States)

    Arantes, Lucas M; Varejão, Eduardo V V; Pelizzaro-Rocha, Karin J; Cereda, Cíntia M S; de Paula, Eneida; Lourenço, Maicon P; Duarte, Hélio A; Fernandes, Sergio A

    2014-05-01

    The aim of this work was to study the interaction between the local anesthetic benzocaine and p-sulfonic acid calix[n]arenes using NMR and theoretical calculations and to assess the effects of complexation on cytotoxicity of benzocaine. The architectures of the complexes were proposed according to (1) H NMR data (Job plot, binding constants, and ROESY) indicating details on the insertion of benzocaine in the cavity of the calix[n]arenes. The proposed inclusion compounds were optimized using the PM3 semiempirical method, and the electronic plus nuclear repulsion energy contributions were performed at the DFT level using the PBE exchange/correlation functional and the 6-311G(d) basis set. The remarkable agreement between experimental and theoretical approaches adds support to their use in the structural characterization of the inclusion complexes. In vitro cytotoxic tests showed that complexation intensifies the intrinsic toxicity of benzocaine, possibly by increasing the water solubility of the anesthetic and favoring its partitioning inside of biomembranes.

  6. An extrapolation scheme for solid-state NMR chemical shift calculations

    Science.gov (United States)

    Nakajima, Takahito

    2017-06-01

    Conventional quantum chemical and solid-state physical approaches include several problems to accurately calculate solid-state nuclear magnetic resonance (NMR) properties. We propose a reliable computational scheme for solid-state NMR chemical shifts using an extrapolation scheme that retains the advantages of these approaches but reduces their disadvantages. Our scheme can satisfactorily yield solid-state NMR magnetic shielding constants. The estimated values have only a small dependence on the low-level density functional theory calculation with the extrapolation scheme. Thus, our approach is efficient because the rough calculation can be performed in the extrapolation scheme.

  7. NMR studies of polysaccharides from brown seaweeds

    Energy Technology Data Exchange (ETDEWEB)

    Noseda, M.D.; Tisher, C.A.; Gorin, P.A.J.; Duarte, M.E.R. [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica; Cerezo, A.S. [Buenos Aires Univ. Nacional (Argentina). Dept. de Quimica Organica

    1997-12-31

    Alginic acid is the major intercellular polysaccharide serving as matrix in the brown algae and is comprised of an unbranched chain of (1->4)-linked {beta}-D-mannuronic acid (M) and {alpha}-L-guluronic acid (G), arranged in a blockwise fashion. The composition of the monomer residues and the block structure varies depending on the source of the polymer. The selective binding of cations to alginate accounts for its ability to form gels, which is dependent on the number and lenght of the G-blocks. They are widely used industrially for their ability to retain water, and for their gelling, viscosifying and stabilizing properties (Smidsrod and draget, 1996). In this study, alginate composition and block structure in Sargassum stenophyllum has been determined by chemical methods and NMR spectroscopic analysis. (author) 4 refs., 3 figs.

  8. Expanding sapphyrin: towards selective phosphate binding.

    Science.gov (United States)

    Katayev, Evgeny A; Boev, Nikolay V; Myshkovskaya, Ekaterina; Khrustalev, Victor N; Ustynyuk, Yu A

    2008-01-01

    The anion-templated syntheses and binding properties of novel macrocyclic oligopyrrole receptors in which pyrrole rings are linked through amide or imine bonds are described. The efficient synthesis was accomplished by anion-templated [1+1] Schiff-base condensation and acylation macrocyclization reactions. Free receptors and their host-guest complexes with hydrochloric acid, acetic acid, tetrabutylammonium chloride, and hydrogen sulfate were analyzed by single-crystal X-ray diffraction analysis. Stability constants with different tetrabutylammonium salts of inorganic acids were determined by standard 1H NMR and UV/Vis titration techniques in [D6]DMSO/0.5% water solution. According to the titration data, receptors containing three pyrrole rings (10 and 12) exhibit high affinity (log Ka=5-7) for bifluoride, acetate, and dihydrogen phosphate, and interact weakly with chloride and hydrogen sulfate. The amido-bipyrrole receptors 11 and 13 with four pyrrole rings exhibit 10(4)- and 10(2)-fold selectivity for dihydrogen phosphate, respectively, as inferred from competitive titrations in the presence of tetrabutylammonium acetate.

  9. NMR monitoring of the SELEX process to confirm enrichment of structured RNA.

    Science.gov (United States)

    Amano, Ryo; Aoki, Kazuteru; Miyakawa, Shin; Nakamura, Yoshikazu; Kozu, Tomoko; Kawai, Gota; Sakamoto, Taiichi

    2017-03-21

    RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 10(15) different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.

  10. Medical applications of NMR imaging and NMR spectroscopy with stable isotopes. Summary

    Energy Technology Data Exchange (ETDEWEB)

    Matwiyoff, N.A.

    1983-01-01

    The current status of NMR imaging and NMR spectroscopy are summarized. For the most part examples from the March 1983 Puerto Rico symposium are used to illustrate the utility of NMR in medicine. 18 refs., 5 figs.

  11. Solution NMR studies on Helicobacter pylori proteins for antibiotic target discovery.

    Science.gov (United States)

    Lee, Ki-Young; Lee, Bong-Jin

    2016-07-01

    Helicobacter pylori (H. pylori) is a well-known widespread pathogenic bacterium that survives in the extremely acidic conditions of the human gastric mucosa. The global prevalence of H. pylori-resistant antibiotics has become an emerging issue in the 21st century and has necessitated the development of novel antibiotic drugs. Many efforts have aimed to discover antibiotic target proteins of H. pylori based on its genome of more than 1600 genes. This article highlights NMR spectroscopy as a valuable tool for determining the structure and dynamics of potential antibiotic-targeted proteins of H. pylori and evaluating their modes of interaction with native or synthetic binding partners. The residue-specific information on binding in solution provides a structural basis to identify and optimize lead compounds. NMR spectroscopy is a powerful method for obtaining details of biomolecular interactions with a broad range of binding affinities. This strength facilitates the identification of the binding interface of the encounter complex that plays an integral role in a variety of biological functions. This low-affinity complex is difficult to crystallize, which impedes structure determination using X-ray crystallography. Additionally, the relative binding affinities can be predicted from the type of spectral change upon binding. High-resolution NMR spectroscopy in combination with advanced computer simulation would provide more confidence in complex structures. The application of NMR to studies of the H. pylori protein could contribute to the development of these targeted novel antibiotics.

  12. Structural Determination of Bis-histidinopeptide Zinc Complexes by 15N NMR (HMBC) Spectra

    Institute of Scientific and Technical Information of China (English)

    ZHOU,Cheng-He; Juan F.Miravet; M.Isabel Burguete; Santiago V.Luis; BAI,Xue; YUAN,Yong

    2004-01-01

    @@ Polynitrogen receptors such as bis-histidine peptides possess strong ability to bind metals, which play much important roles in medicinal, bioinorganic, bioorganic, biomimetic and supramolecular chemistry. In order to investigate the interaction of these hosts with a variety of neutral, cationic and anionic guests, several techniques, for example, NMR,potentiometric tirations and monocrystal X-ray diffraction have been employed. Among them NMR is a powerful technique for unraveling the structure of polynitrogen receptors as long as they are in solution where the rapid tumbling of molecules averages out the anisotropies such as chemical shift and dipole-dipole interactions. General 1H NMR approach has been widely used for the study of host-guest interaction, but it is difficult for the accurate measurement in complexes structures, particularly metal complexes structures in which how the polynitrogen receptors bind metal, and which nitrogen binds metal and so on.

  13. Measurable values, numbers and fundamental physical constants: Is the Boltzmann constant Kb a fundamental physical constant?

    Directory of Open Access Journals (Sweden)

    Bormashenko Edward

    2009-01-01

    Full Text Available The status of fundamental physical constants is discussed. The nature of fundamental physical constants is cleared up, based on the analysis of the Boltzmann constant. A new definition of measurable values, 'mathematical' and 'physical' numbers and fundamental physical constants is proposed. Mathematical numbers are defined as values insensitive to the choice of both units and frames of reference, whereas 'physical numbers' are dimensionless values, insensitive to transformations of units and sensitive to the transformations of the frames of reference. Fundamental constants are classified as values sensitive to transformations of the units and insensitive to transformations of the frames of reference. It is supposed that a fundamental physical constant necessarily allows diminishing the number of independent etalons in a system of units.

  14. α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF.

    Science.gov (United States)

    Guo, Ming; Wang, Xiaomeng; Lu, Xiaowang; Wang, Hongzheng; Brodelius, Peter E

    2016-01-01

    In order to obtain the biological active compound, α-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of α-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of α-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (1H NMR and 13C NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained α-mangostin was 95.6% as determined by HPLC analysis. The binding of native α-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that α-mangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to α-mangostin is 6.4832×105 L/mol and 1.4652×105 L/mol at 298 K and 7.8619×105 L/mol and 1.1582×105 L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between α-mangostin and HSA or TRF were also obtained by virtue of the Förster theory of non-radiation energy transfer. The effect of α-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between α-mangostin and HSA is hydrophobic interactions, while the main interaction between α-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.

  15. Spectrophotometric determination of association constant

    DEFF Research Database (Denmark)

    2016-01-01

    Least-squares 'Systematic Trial-and-Error Procedure' (STEP) for spectrophotometric evaluation of association constant (equilibrium constant) K and molar absorption coefficient E for a 1:1 molecular complex, A + B = C, with error analysis according to Conrow et al. (1964). An analysis of the Charg...

  16. Constant-Pressure Hydraulic Pump

    Science.gov (United States)

    Galloway, C. W.

    1982-01-01

    Constant output pressure in gas-driven hydraulic pump would be assured in new design for gas-to-hydraulic power converter. With a force-multiplying ring attached to gas piston, expanding gas would apply constant force on hydraulic piston even though gas pressure drops. As a result, pressure of hydraulic fluid remains steady, and power output of the pump does not vary.

  17. Spectrophotometric determination of association constant

    DEFF Research Database (Denmark)

    Spanget-Larsen, Jens

    2016-01-01

    Least-squares 'Systematic Trial-and-Error Procedure' (STEP) for spectrophotometric evaluation of association constant (equilibrium constant) K and molar absorption coefficient E for a 1:1 molecular complex, A + B = C, with error analysis according to Conrow et al. (1964). An analysis of the Charge...

  18. NMR Dynamic Studies in Living Systems

    Institute of Scientific and Technical Information of China (English)

    闫永彬; 范明杰; 罗雪春; 张日清

    2002-01-01

    Nuclear magnetic resonance (NMR) can noninvasively monitor the intracellular concentrations and kinetic properties of numerous inorganic and organic compounds. These characteristics have made NMR a useful tool for dynamic studies of living systems. Applications of NMR to living systems have successfully extended to many areas, including studies of metabolic regulation, ion transport, and intracellular reaction rates in vivo. The major purpose of this review is to summarize the results that can be obtained by modern NMR techniques in living systems. With the advances of new techniques, NMR measurements of various nuclides have been performed for specific physiological purposes. Although some technical problems still remain and there are still discrepancies between NMR and traditional biochemical results, the abundant and unique information obtained from NMR spectra suggests that NMR will be more extensively applied in future studies of living systems. The fast development of these new techniques is providing many new NMR applications in living systems, as well as in structural biology.

  19. Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity† †Electronic supplementary information (ESI) available: NMR chemical shift characterisation of modified heparins, protein sequence alignment methodology and data, protein binding and activity assay dose-response curves. See DOI: 10.1039/c6mb00432f Click here for additional data file.

    Science.gov (United States)

    Ahmed, Yassir A.; Yates, Edwin A.; Moss, Diana J.; Loeven, Markus A.; Hussain, Sadaf-Ahmahni; Hohenester, Erhard; Turnbull, Jeremy E.

    2016-01-01

    Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit–Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo. PMID:27502551

  20. On the configuration of five-membered rings: a spin-spin coupling constant approach.

    Science.gov (United States)

    Napolitano, José G; Gavín, José A; García, Celina; Norte, Manuel; Fernández, José J; Daranas, Antonio Hernández

    2011-05-27

    Five-membered rings are clearly among the most common structural motifs found in chemistry and biology. Nevertheless, the configuration of conformationally mobile five-membered rings is often difficult to assign from nuclear magnetic resonance (NMR) data. A simple, reliable, and efficient approach for the stereochemical analysis of five-membered rings based on the measurement of NMR coupling constants is presented. Density functional theory calculations using representative conformations of the full conformational space available to rings with different substitution patterns were used to identify differences between the accessible coupling constant values for cis and trans relative orientations of the substituents. The calculations were assessed experimentally using NMR data obtained from a number of models. This approach can be easily used to analyze different five-membered rings, such as oxolanes, cyclopentanes, furanosides and pyrrolidines, and their relative configuration can be determined without the need for making further conformational considerations.

  1. Cu(I) complexes of bis(methyl)(thia/selena) salen ligands: Synthesis, characterization, redox behavior and DNA binding studies

    Science.gov (United States)

    Asatkar, Ashish K.; Tripathi, Mamta; Panda, Snigdha; Pande, Rama; Zade, Sanjio S.

    2017-01-01

    Mononuclear cuprous complexes 1 and 2, [{CH3E(o-C6H4)CH = NCH2}2Cu]ClO4; E = S/Se, have been synthesized by the reaction of bis(methyl)(thia/selena) salen ligands and [Cu(CH3CN)4]ClO4. Both the products were characterized by elemental analysis, ESI-MS, FT-IR, 1H/13C/77Se NMR, and cyclic voltammetry. The complexes possess tetrahedral geometry around metal center with the N2S2/N2Se2 coordination core. Cyclic voltammograms of complexes 1 and 2 displayed reversible anodic waves at E1/2 = + 0.08 V and + 0.10 V, respectively, corresponding to the Cu(I)/Cu(II) redox couple. DNA binding studies of both the complexes were performed applying absorbance, fluorescence and molecular docking techniques. Competitive binding experiment of complexes with ct-DNA against ethidium bromide is performed to predict the mode of binding. The results indicate the groove binding mode of complexes 1 and 2 to DNA. The binding constants revealed the strong binding affinity of complexes towards ct-DNA.

  2. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  3. NMR studies of actinide dioxides

    Energy Technology Data Exchange (ETDEWEB)

    Tokunaga, Y. [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan)], E-mail: tokunaga.yo@jaea.go.jp; Sakai, H.; Fujimoto, T.; Kambe, S.; Walstedt, R.E.; Ikushima, K.; Yasuoka, H. [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Aoki, D.; Homma, Y. [Institute for Materials Research, Tohoku University, Oarai, Ibaraki 311-1313 (Japan); Haga, Y.; Matsuda, T.D.; Ikeda, S.; Yamamoto, E.; Nakamura, A. [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Shiokawa, Y. [Institute for Materials Research, Tohoku University, Oarai, Ibaraki 311-1313 (Japan); Nakajima, K.; Arai, Y. [Department of Nuclear Energy System, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Onuki, Y. [Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195 (Japan); Department of Physics, Osaka University, Toyonaka, Osaka 560-0043 (Japan)

    2007-10-11

    {sup 17}O NMR measurements have been performed on a series of the actinide dioxides, UO{sub 2}, NpO{sub 2} and PuO{sub 2}. Although the {sup 17}O NMR spectra in these materials are similar at higher temperatures, the low-temperature spectra present are significantly different. In UO{sub 2} we have observed a wide spectrum, forming a rectangular shape below T{sub N}=30 K. In NpO{sub 2}, on the other hand, the spectra broaden rather gradually and exhibit a two-peak structure below T{sub 0}=26 K. In PuO{sub 2}, neither spectrum broadening nor splitting has been observed. We show that these NMR spectra clearly indicate the different nature of the low-temperature magnetic ground states in these actinide compounds.

  4. Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy.

    Science.gov (United States)

    Adams, Kristie M; Marzilli, Patricia A; Marzilli, Luigi G

    2007-10-29

    Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination

  5. NMR and dynamics of biopolymers

    Energy Technology Data Exchange (ETDEWEB)

    Lian, L.Y.; Barsukov, I.L. [Leicester Univ. (United Kingdom)

    1994-12-31

    Several basic experimental analytical NMR techniques that are frequently used for the qualitative and quantitative analysis of dynamic and exchange processes, focusing on proteins systems, are described: chemical exchange (slow exchange, fast exchange, intermediate exchange), heteronuclear relaxation measurements (relaxation parameters, strategy of relaxation data analysis, experimental results and examples, motional model interpretation of relaxation data, homonuclear relaxation); slow large-scale exchange and hydrogen-deuterium exchange are also studied: mechanisms of hydrogen exchange in a native protein, methods for measuring amide exchange rates by NMR, interpretation of amide exchange rates. 9 fig., 3 tab., 56 ref.

  6. Spectral Estimation of NMR Relaxation

    Science.gov (United States)

    Naugler, David G.; Cushley, Robert J.

    2000-08-01

    In this paper, spectral estimation of NMR relaxation is constructed as an extension of Fourier Transform (FT) theory as it is practiced in NMR or MRI, where multidimensional FT theory is used. nD NMR strives to separate overlapping resonances, so the treatment given here deals primarily with monoexponential decay. In the domain of real error, it is shown how optimal estimation based on prior knowledge can be derived. Assuming small Gaussian error, the estimation variance and bias are derived. Minimum bias and minimum variance are shown to be contradictory experimental design objectives. The analytical continuation of spectral estimation is constructed in an optimal manner. An important property of spectral estimation is that it is phase invariant. Hence, hypercomplex data storage is unnecessary. It is shown that, under reasonable assumptions, spectral estimation is unbiased in the context of complex error and its variance is reduced because the modulus of the whole signal is used. Because of phase invariance, the labor of phasing and any error due to imperfect phase can be avoided. A comparison of spectral estimation with nonlinear least squares (NLS) estimation is made analytically and with numerical examples. Compared to conventional sampling for NLS estimation, spectral estimation would typically provide estimation values of comparable precision in one-quarter to one-tenth of the spectrometer time when S/N is high. When S/N is low, the time saved can be used for signal averaging at the sampled points to give better precision. NLS typically provides one estimate at a time, whereas spectral estimation is inherently parallel. The frequency dimensions of conventional nD FT NMR may be denoted D1, D2, etc. As an extension of nD FT NMR, one can view spectral estimation of NMR relaxation as an extension into the zeroth dimension. In nD NMR, the information content of a spectrum can be extracted as a set of n-tuples (ω1, … ωn), corresponding to the peak maxima

  7. Unraveling the meaning of chemical shifts in protein NMR.

    Science.gov (United States)

    Berjanskii, Mark V; Wishart, David S

    2017-07-15

    Chemical shifts are among the most informative parameters in protein NMR. They provide wealth of information about protein secondary and tertiary structure, protein flexibility, and protein-ligand binding. In this report, we review the progress in interpreting and utilizing protein chemical shifts that has occurred over the past 25years, with a particular focus on the large body of work arising from our group and other Canadian NMR laboratories. More specifically, this review focuses on describing, assessing, and providing some historical context for various chemical shift-based methods to: (1) determine protein secondary and super-secondary structure; (2) derive protein torsion angles; (3) assess protein flexibility; (4) predict residue accessible surface area; (5) refine 3D protein structures; (6) determine 3D protein structures and (7) characterize intrinsically disordered proteins. This review also briefly covers some of the methods that we previously developed to predict chemical shifts from 3D protein structures and/or protein sequence data. It is hoped that this review will help to increase awareness of the considerable utility of NMR chemical shifts in structural biology and facilitate more widespread adoption of chemical-shift based methods by the NMR spectroscopists, structural biologists, protein biophysicists, and biochemists worldwide. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017. Published by Elsevier B.V.

  8. DETERMINATION OF STABILITY CONSTANTS OF MANGANESE (II ...

    African Journals Online (AJOL)

    DR. AMINU

    Keywords: Amino acids, dissociation constant, potentiometry, stability constant. INTRODUCTION ... constants of manganese (II) amino acid complexes using potentiometer. .... Principles of Biochemistry Third Edition,. Worth publishers, 41 ...

  9. Thermostatted micro-reactor NMR probe head for monitoring fast reactions

    Science.gov (United States)

    Brächer, A.; Hoch, S.; Albert, K.; Kost, H. J.; Werner, B.; von Harbou, E.; Hasse, H.

    2014-05-01

    A novel nuclear magnetic resonance (NMR) probe head for monitoring fast chemical reactions is described. It combines micro-reaction technology with capillary flow NMR spectroscopy. Two reactants are fed separately into the probe head where they are effectively mixed in a micro-mixer. The mixed reactants then pass through a capillary NMR flow cell that is equipped with a solenoidal radiofrequency coil where the NMR signal is acquired. The whole flow path of the reactants is thermostatted using the liquid FC-43 (perfluorotributylamine) so that exothermic and endothermic reactions can be studied under almost isothermal conditions. The set-up enables kinetic investigation of reactions with time constants of only a few seconds. Non-reactive mixing experiments carried out with the new probe head demonstrate that it facilitates the acquisition of constant highly resolved NMR signals suitable for quantification of different species in technical mixtures. Reaction kinetic measurements on a test system are presented that prove the applicability of the novel NMR probe head for monitoring fast reactions.

  10. Thermostatted micro-reactor NMR probe head for monitoring fast reactions.

    Science.gov (United States)

    Brächer, A; Hoch, S; Albert, K; Kost, H J; Werner, B; von Harbou, E; Hasse, H

    2014-05-01

    A novel nuclear magnetic resonance (NMR) probe head for monitoring fast chemical reactions is described. It combines micro-reaction technology with capillary flow NMR spectroscopy. Two reactants are fed separately into the probe head where they are effectively mixed in a micro-mixer. The mixed reactants then pass through a capillary NMR flow cell that is equipped with a solenoidal radiofrequency coil where the NMR signal is acquired. The whole flow path of the reactants is thermostatted using the liquid FC-43 (perfluorotributylamine) so that exothermic and endothermic reactions can be studied under almost isothermal conditions. The set-up enables kinetic investigation of reactions with time constants of only a few seconds. Non-reactive mixing experiments carried out with the new probe head demonstrate that it facilitates the acquisition of constant highly resolved NMR signals suitable for quantification of different species in technical mixtures. Reaction kinetic measurements on a test system are presented that prove the applicability of the novel NMR probe head for monitoring fast reactions.

  11. Varying constants, Gravitation and Cosmology

    CERN Document Server

    Uzan, Jean-Philippe

    2010-01-01

    Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. It is thus of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We thus detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, Solar system observations, meteorites dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describ...

  12. Ruzsa's Constant on Additive Functions

    Institute of Scientific and Technical Information of China (English)

    Jin Hui FANG; Yong Gao CHEN

    2013-01-01

    A function f:N → R is called additive if f(mn) =f(m)+f(n) for all m,n with (m,n) =1.Let μ(x) =maxn≤x(f(n)-f(n + 1)) and v(x) =maxn≤x(f(n + 1)-f(n)).In 1979,Ruzsa proved that there exists a constant c such that for any additive function f,μ(x) ≤ cv(x2) + cf,where cf is a constant depending only on f.Denote by Raf the least such constant c.We call Raf Ruzsa's constant on additive functions.In this paper,we prove that Raf ≤ 20.

  13. Naturally Time Dependent Cosmological Constant

    CERN Document Server

    Gregori, A

    2004-01-01

    In the light of the proposal of hep-th/0207195, we discuss in detail the issue of the cosmological constant, explaining how can string theory naturally predict the value which is experimentally observed, without low-energy supersymmetry.

  14. Inflation and the cosmological constant

    Directory of Open Access Journals (Sweden)

    FENG Chaojun

    2014-08-01

    Full Text Available By assuming the cosmological “constant” is no longer a constant during the inflation epoch,it is found that the cosmological constant fine-tuning problem is solved.In the meanwhile,inflation models could predict a large tensor-to-scalar ratio,correct power spectral index and a larger running of it.Furthermore,the e-folding number is large enough to overcome the horizon,flatness problems in the Big Bang cosmology.

  15. Push-through Direction Injectin NMR Automation

    Science.gov (United States)

    Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are the two major spectroscopic techniques successfully used in metabolomics studies. The non-invasive, quantitative and reproducible characteristics make NMR spectroscopy an excellent technique for detection of endogeno...

  16. Push-through Direction Injectin NMR Automation

    Science.gov (United States)

    Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are the two major spectroscopic techniques successfully used in metabolomics studies. The non-invasive, quantitative and reproducible characteristics make NMR spectroscopy an excellent technique for detection of endogeno...

  17. "Solvent Effects" in 1H NMR Spectroscopy.

    Science.gov (United States)

    Cavaleiro, Jose A. S.

    1987-01-01

    Describes a simple undergraduate experiment in chemistry dealing with the "solvent effects" in nuclear magnetic resonance (NMR) spectroscopy. Stresses the importance of having students learn NMR spectroscopy as a tool in analytical chemistry. (TW)

  18. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  19. Binding of reactive organophosphate by oximes via hydrogen bond

    Indian Academy of Sciences (India)

    Andrea Pappalardo; Maria E Amato; Francesco P Ballistreri; Valentina La Paglia Fragola; Gaetano A Tomaselli; Rosa Maria Toscano; Giuseppe Trusso Sfrazzetto

    2013-07-01

    In this contribution, the ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments confirm that oximes bind the organophosphate via hydrogen bond.

  20. Varying Constants, Gravitation and Cosmology

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Uzan

    2011-03-01

    Full Text Available Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. Thus, it is of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, solar system observations, meteorite dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describe the main theoretical frameworks in which the low-energy constants may actually be varying and we focus on the unification mechanisms and the relations between the variation of different constants. To finish, we discuss the more speculative possibility of understanding their numerical values and the apparent fine-tuning that they confront us with.

  1. Varying Constants, Gravitation and Cosmology.

    Science.gov (United States)

    Uzan, Jean-Philippe

    2011-01-01

    Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. Thus, it is of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, solar system observations, meteorite dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describe the main theoretical frameworks in which the low-energy constants may actually be varying and we focus on the unification mechanisms and the relations between the variation of different constants. To finish, we discuss the more speculative possibility of understanding their numerical values and the apparent fine-tuning that they confront us with.

  2. Varying Constants, Gravitation and Cosmology

    Science.gov (United States)

    Uzan, Jean-Philippe

    2011-12-01

    Fundamental constants are a cornerstone of our physical laws. Any constant varying in space and/or time would reflect the existence of an almost massless field that couples to matter. This will induce a violation of the universality of free fall. Thus, it is of utmost importance for our understanding of gravity and of the domain of validity of general relativity to test for their constancy. We detail the relations between the constants, the tests of the local position invariance and of the universality of free fall. We then review the main experimental and observational constraints that have been obtained from atomic clocks, the Oklo phenomenon, solar system observations, meteorite dating, quasar absorption spectra, stellar physics, pulsar timing, the cosmic microwave background and big bang nucleosynthesis. At each step we describe the basics of each system, its dependence with respect to the constants, the known systematic effects and the most recent constraints that have been obtained. We then describe the main theoretical frameworks in which the low-energy constants may actually be varying and we focus on the unification mechanisms and the relations between the variation of different constants. To finish, we discuss the more speculative possibility of understanding their numerical values and the apparent fine-tuning that they confront us with.

  3. Synthesis and DNA-binding study of imidazole linked thiazolidinone derivatives.

    Science.gov (United States)

    War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

    2017-02-01

    A novel series of imidazole-linked thiazolidinone hybrid molecules were designed and synthesized through a feasible synthetic protocol. The molecules were characterized with Fourier transform infrared (FT-IR), (1) H nuclear magnetic resonance (NMR), (13) C NMR and high-resolution mass spectrometry (HRMS) techniques. In vitro susceptibility tests against Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. aeruginosa) gave highly promising results. The most active molecule (3e) gave a minimal inhibitory concentration (MIC) value of 3.125 μg/mL which is on par with the reference drug streptomycin. Structure-activity relationships revealed activity enhancement by nitro and chloro groups when they occupied meta position of the arylidene ring in 2-((3-(imidazol-1-yl)propyl)amino)-5-benzylidenethiazolidin-4-ones. DNA-binding study of the most potent molecule 3e with salmon milt DNA (sm-DNA) under simulated physiological pH was probed with UV-visible absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that compound 3e has a strong affinity towards DNA and binds at DNA minor groove with a binding constant (Kb ) 0.18 × 10(2)  L mol(-1) . Molecular docking simulations predicted strong affinity of 3e towards DNA with a binding affinity (ΔG) -8.5 kcal/mol. Van der Waals forces, hydrogen bonding and hydrophobic interactions were predicted as the main forces of interaction. The molecule 3e exhibited specific affinity towards adenine-thiamine base pairs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  4. (19)F NMR spectroscopic characterization of the interaction of niflumic acid with human serum albumin.

    Science.gov (United States)

    Kitamura, Keisuke; Omran, Ahmed A; Takegami, Shigehiko; Tanaka, Rumi; Kitade, Tatsuya

    2007-04-01

    The interaction of a non-steroidal anti-inflammatory drug, niflumic acid (NFA), with human serum albumin (HSA) has been investigated by (19)F nuclear magnetic resonance (NMR) spectroscopy. A (19)F NMR spectrum of NFA in a buffered (pH 7.4) solution of NaCl (0.1 mol L(-1)) contained a single sharp signal of its CF(3) group 14.33 ppm from the internal reference 2,2,2-trifluoroethanol. Addition of 0.6 mmol L(-1) HSA to the NFA buffer solution caused splitting of the CF(3) signal into two broadened signals, shifted to the lower fields of 14.56 and 15.06 ppm, with an approximate intensity ratio of 1:3. Denaturation of HSA by addition of 3.0 mol L(-1) guanidine hydrochloride (GU) restored a single sharp signal of CF(3) at 14.38 ppm, indicating complete liberation of NFA from HSA as a result of its denaturation. These results suggest that the binding is reversible and occurs in at least two HSA regions. Competitive (19)F NMR experiments using warfarin, dansyl-L: -asparagine, and benzocaine (site I ligands), and L: -tryptophan and ibuprofen (site II ligands) revealed that NFA binds to site I at two different regions, Ia and Ib, in the ratio 1:3. By use of (19)F NMR with NFA as an (19)F NMR probe the nonfluorinated site I-binding drugs sulfobromophthalein and iophenoxic acid were also found to bind sites Ia and Ib, respectively. These results illustrate the usefulness and convenience of (19)F NMR for investigation of the HSA binding of both fluorinated and nonfluorinated drugs.

  5. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  6. High resolution NMR theory and chemical applications

    CERN Document Server

    Becker, Edwin D

    1969-01-01

    High Resolution NMR: Theory and Chemical Applications focuses on the applications of nuclear magnetic resonance (NMR), as well as chemical shifts, lattices, and couplings. The book first offers information on the theory of NMR, including nuclear spin and magnetic moment, spin lattice relaxation, line widths, saturation, quantum mechanical description of NMR, and ringing. The text then ponders on instrumentation and techniques and chemical shifts. Discussions focus on the origin of chemical shifts, reference compounds, empirical correlations of chemical shifts, modulation and phase detection,

  7. NMR detection of slow conformational dynamics in an endonuclease toxin

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, Sara B.-M.; Boetzel, Ruth; MacDonald, Colin [University of East Anglia, School of Chemical Sciences (United Kingdom); Lian Luyun [Leicester University, Biological NMR Centre (United Kingdom); Pommer, Ansgar J. [University of East Anglia, School of Biological Sciences (United Kingdom); Reilly, Ann; James, Richard; Kleanthous, Colin [Leicester University, Biological NMR Centre (United Kingdom); Moore, Geoffrey R. [University of East Anglia, School of Chemical Sciences (United Kingdom)

    1998-07-15

    The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid{sup 15} N- and {sup 13}C/{sup 15}N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro{sup 65}-Asn{sup 72}, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D {sup 1}H-{sup 1}H-{sup 15}N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C{sup {alpha}} and C{sup {beta}} chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp{sup 22} or Trp{sup 58}, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D{sup 1} H-{sup 1}H-{sup 15}N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, N{sub A} and N{sub B}, which interchange at 15 {sup o}C with a forward rate constant of 1.61 {+-} 0.5 s{sup -1} and a backward rate constant of 1.05 {+-} 0.5 s{sup -1}. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743-13750 and 13751-13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the N{sub A} and N{sub B

  8. Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

    OpenAIRE

    Shoshan, Michal S.; Tshuva, Edit Y.; Shalev, Deborah E.

    2013-01-01

    Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.

  9. Time domain NMR applied to food products

    NARCIS (Netherlands)

    Duynhoven, van J.P.M.; Voda, A.; Witek, M.M.; As, van H.

    2010-01-01

    Time-domain NMR is being used throughout all areas of food science and technology. A wide range of one- and two-dimensional relaxometric and diffusometric applications have been implemented on cost-effective, robust and easy-to-use benchtop NMR equipment. Time-domain NMR applications do not only

  10. $^{31}$Mg $\\beta$-NMR applied in chemistry and biochemistry

    CERN Multimedia

    Magnesium ions, Mg$^{2+}$, are essential in biological systems, taking part in practically all phosphate chemistry, in photosynthesis as an integral component of chlorophyll, and they are regulated via transport through selective membrane proteins. Nonetheless, the function of magnesium ions in biochemistry is difficult to characterize, as it is practically invisible to current experimental techniques. With this proposal we aim to advance the use of $^{31}$Mg $\\beta$-NMR to liquid samples, building on the experience from the successful Letter of Intent INTC-I-088 “$\\beta$-NMR as a novel technique for biological applications”. Initially a series of experiments will be conducted aiming to characterize the coordination chemistry of Mg$^{2+}$ in ionic liquids (ILs), demonstrating that it is possible within the lifetime of the radioisotope to achieve binding of Mg$^{2+}$ to a molecule dissolved in the IL. ILs are chosen as they display a very low vapor pressure, and are thus straightforwardly compatible with t...

  11. Development and Investigation of NMR tools for chiral compound identification

    Energy Technology Data Exchange (ETDEWEB)

    Alam, Todd Michael [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Electronic, Optical and Nano Materials; Lansdon, Rick [Oak Ridge Inst. for Science and Education (ORISE), Oak Ridge, TN (United States)

    2014-09-01

    The goal behind the assigned summer project was to investigate the ability of nuclear magnetic resonance spectroscopy (NMR) to identify enantiomers of select chiral organo-fluorophosphates (OFPs) compounds which are analogs of chemical warfare agents (CWAs, e.g. Sarin). This involved investigations utilizing chiral solvating agents (CSAs) and characterizing the binding phenomena with cyclodextrins. The resolution of OFPs enantiomers using NMR would be useful for research into toxicodynamics and toxicokinetics in biological systems due to the widely differing properties of the CWA enantiomers [1]. The optimization of decontamination abilities in the case of a CWA events, with this method’s potential rapidity and robustness, as well as the development of models correlating chiral compounds with CSAs for optimal resolution are all rational benefits of this research.

  12. Exploring the DNA binding mode of transition metal based biologically active compounds

    Science.gov (United States)

    Raman, N.; Sobha, S.

    2012-01-01

    Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV-vis, 1H NMR and EPR spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spectroscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with DNA was also studied by molecular modeling with special reference to docking. The experimental and docking results revealed that the complexes have the ability of interaction with DNA of minor groove binding mode. The intrinsic binding constants ( Kb) of the complexes with CT-DNA were found out which show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pUC19 DNA in the presence of AH 2 (ascorbic acid). Moreover, the oxidative cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free ligands.

  13. Novel Monomeric Phenanthroline—Thallium(Ⅲ) Complexes Multinuclear NMR Characterization in Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    GuiBinMA; JuliusGLASER

    2002-01-01

    A novel complex of monomeric thallium (Ⅲ) with the nitrogen donor ligand phenanthroline (phen) has been prepared and characterized by multimuclear NMR(1H,13C,205Tl). The three complexes exist in equilibria in DMSO and acetonitrile solution, which was proved by the 205Tl NMR spectra. The 1H and 13C NMR spectra of tris-phen T1(Ⅲ) complex have been measured, where the spin-spin coupling between T1(I=1/2) and 13C or 1H signals were observed with the 1H and 13C NMR spectroscopy in acetonitrile. The coupling constants are presented and the chemical shifts of complexes are discussed in detail.

  14. Novel Monomeric Phenanthroline - Thallium(Ⅲ) Complexes Multinuclear NMR Characterization in Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A novel complex of monomeric thallium(III) with the nitrogen donor ligand phenanthrolinc (phen) has been prepared and characterized by multinuclear NMR (1H, 13C, 205T1).The three complexes exist in equilibria in DMSO and acetonitrile solution, which was proved by the 205Tl NMR spectra. The 1H and 13C NMR spectra of tris-phen Tl(III) complex have been measured, where the spin-spin coupling between TI (1 = 1/2) and 13C or 1H signals were observed with the 1H and 13C NMR spectroscopy in acetonitrile. The coupling constants are presented and the chemical shifts of complexes are discussed in detail.

  15. The characterization of NMR signal for blood pressure monitoring system and its testing

    Directory of Open Access Journals (Sweden)

    Bambang Murdaka Eka Jati

    2016-02-01

    Full Text Available ABSTRACT A blood monitoring system based on NMR method has been designed on constructed. This set-up of equipment used magnetic permanent, radio frequency (RF, receiver coil (RC, function generator (FG, amplifier which included the filter, as well as the oscilloscope digital storage. The background of this research was based on the sensitivity of NMR signal. The signal must be separated from signals background. This method was done by adjusting the frequency on FG, which was connected to radio frequency (RF coil, on empty sample. Subsequently, NMR signal was received by RC, and that signal could be shown on oscilloscope at resonance condition. The true frequency on NMR signal was Larmor frequency, and the other was background. The two variables of this experiment were the position of RF coil and the location temperature (20 up to 30oC. In conclusion, the resonance frequency of NMR signal (as Larmor frequency was 4.7 MHz (at static magnetic field of 1,600 gauss and it could be separated from background signals (3.4 and 6.2 MHz, and that signal was almost constant to room temperature. The equipment was used for sample testing. It gave systole/diastole data of 110/70 mmHg (on sphygmomanometer that was similar to 17/9 mV (on NMR signal. ABSTRAK Telah dikembangkan alat pemantauan tekanan darah berdasar prinsip NMR.

  16. Solid State NMR Studies of Energy Conversion and Storage Materials

    Science.gov (United States)

    Jankuru Hennadige, Sohan Roshel De Silva

    NMR (Nuclear magnetic resonance) spectroscopy is utilized to study energy conversion and storage materials. Different types of NMR techniques including Magic Angle Spinning, Cross-polarization and relaxation measurement experiments were employed. Four different projects are discussed in this dissertation. First, three types of CFx battery materials were investigated. Electrochemical studies have demonstrated different electrochemical performances by one type, delivering superior performance over the other two. 13C and 19F MAS NMR techniques are employed to identify the atomic/molecular structural factors that might account for differences in electrochemical performance among different types. Next as the second project, layered polymer dielectrics were investigated by NMR. Previous studies have shown that thin film capacitors are improved by using alternate layers of two polymers with complementary properties: one with a high breakdown strength and one with high dielectric constant as opposed to monolithic layers. 13C to 1H cross-polarization techniques were used to investigate any inter-layer properties that may cause the increase in the dielectric strength. The third project was to study two types of thermoelectric materials. These samples were made of heavily doped phosphorous and boron in silicon by two different methods: ball-milled and annealed. These samples were investigated by NMR to determine the degree of disorder and obtain insight into the doping efficiency. The last ongoing project is on a lithium-ion battery system. The nature of passivating layers or the solid electrolyte interphase (SEI) formed on the electrodes surface is important because of the direct correlation between the SEI and the battery life time/durability. Multinuclear (7Li, 19F, 31P) techniques are employed to identify the composition of the SEI formation of both positive and negative electrodes.

  17. Learning Read-constant Polynomials of Constant Degree modulo Composites

    DEFF Research Database (Denmark)

    Chattopadhyay, Arkadev; Gavaldá, Richard; Hansen, Kristoffer Arnsfelt;

    2011-01-01

    Boolean functions that have constant degree polynomial representation over a fixed finite ring form a natural and strict subclass of the complexity class \\textACC0ACC0. They are also precisely the functions computable efficiently by programs over fixed and finite nilpotent groups. This class...... is not known to be learnable in any reasonable learning model. In this paper, we provide a deterministic polynomial time algorithm for learning Boolean functions represented by polynomials of constant degree over arbitrary finite rings from membership queries, with the additional constraint that each variable...

  18. Effective cosmological constant induced by stochastic fluctuations of Newton's constant

    Science.gov (United States)

    de Cesare, Marco; Lizzi, Fedele; Sakellariadou, Mairi

    2016-09-01

    We consider implications of the microscopic dynamics of spacetime for the evolution of cosmological models. We argue that quantum geometry effects may lead to stochastic fluctuations of the gravitational constant, which is thus considered as a macroscopic effective dynamical quantity. Consistency with Riemannian geometry entails the presence of a time-dependent dark energy term in the modified field equations, which can be expressed in terms of the dynamical gravitational constant. We suggest that the late-time accelerated expansion of the Universe may be ascribed to quantum fluctuations in the geometry of spacetime rather than the vacuum energy from the matter sector.

  19. Effective cosmological constant induced by stochastic fluctuations of Newton's constant

    CERN Document Server

    de Cesare, Marco; Sakellariadou, Mairi

    2016-01-01

    We consider implications of the microscopic dynamics of spacetime for the evolution of cosmological models. We argue that quantum geometry effects may lead to stochastic fluctuations of the gravitational constant, which is thus considered as a macroscopic effective dynamical quantity. Consistency with Riemannian geometry entails the presence of a time-dependent dark energy term in the modified field equations, which can be expressed in terms of the dynamical gravitational constant. We suggest that the late-time accelerated expansion of the Universe may be ascribed to quantum fluctuations in the geometry of spacetime rather than the vacuum energy from the matter sector.

  20. Two-dimensional NMR exchange spectroscopy. Quantitative treatment of multisite exchanging systems

    Science.gov (United States)

    Abel, Edward W.; Coston, Timothy P. J.; Orrell, Keith G.; Šik, Vladimir; Stephenson, David

    A general method for evaluating rate constants in complex exchange networks with N-sites from two-dimensional EXSY (NOESY) NMR spectra is proposed. A computer program D2DNMR capable of performing signal intensity to exchange rate calculations (and vice versa), based on a matrix formalism, is outlined. The method is illustrated by 195Pt 2D NMR studies of the A ⇌ B ⇌ C spin system arising from pyramidal sulfur inversion in platinum(IV) complexes of type [Pt XMe 3(MeSCH 2CH 2SMe)] ( X = Cl, I). Comparison with 1H NMR bandshape analyses of the same compounds shows high agreement between the rate constants and activation parameters determined by both techniques. Mechanisms of 195Pt spin-lattice relaxation are briefly discussed.

  1. Dissipative particle dynamics of diffusion-NMR requires high Schmidt-numbers

    Science.gov (United States)

    Azhar, Mueed; Greiner, Andreas; Korvink, Jan G.; Kauzlarić, David

    2016-06-01

    We present an efficient mesoscale model to simulate the diffusion measurement with nuclear magnetic resonance (NMR). On the level of mesoscopic thermal motion of fluid particles, we couple the Bloch equations with dissipative particle dynamics (DPD). Thereby we establish a physically consistent scaling relation between the diffusion constant measured for DPD-particles and the diffusion constant of a real fluid. The latter is based on a splitting into a centre-of-mass contribution represented by DPD, and an internal contribution which is not resolved in the DPD-level of description. As a consequence, simulating the centre-of-mass contribution with DPD requires high Schmidt numbers. After a verification for fundamental pulse sequences, we apply the NMR-DPD method to NMR diffusion measurements of anisotropic fluids, and of fluids restricted by walls of microfluidic channels. For the latter, the free diffusion and the localisation regime are considered.

  2. Computer-aided design of fragment mixtures for NMR-based screening.

    Directory of Open Access Journals (Sweden)

    Xavier Arroyo

    Full Text Available Fragment-based drug discovery is widely applied both in industrial and in academic screening programs. Several screening techniques rely on NMR to detect binding of a fragment to a target. NMR-based methods are among the most sensitive techniques and have the further advantage of yielding a low rate of false positives and negatives. However, NMR is intrinsically slower than other screening techniques; thus, to increase throughput in NMR-based screening, researchers often assay mixtures of fragments, rather than single fragments. Herein we present a fast and straightforward computer-aided method to design mixtures of fragments taken from a library that have minimized NMR signal overlap. This approach enables direct identification of one or several active fragments without the need for deconvolution. Our approach entails encoding of NMR spectra into a computer-readable format that we call a fingerprint, and minimizing the global signal overlap through a Monte Carlo algorithm. The scoring function used favors a homogenous distribution of the global signal overlap. The method does not require additional experimental work: the only data required are NMR spectra, which are generally recorded for each compound as a quality control measure before its insertion into the library.

  3. Ultrasensitive anion detection by NMR spectroscopy: a supramolecular strategy based on modulation of chemical exchange rate.

    Science.gov (United States)

    Perruchoud, Loïse H; Hadzovic, Alen; Zhang, Xiao-An

    2015-06-08

    NMR spectroscopy is a powerful tool for monitoring molecular interactions and is widely used to characterize supramolecular systems at the atomic level. NMR is limited for sensing purposes, however, due to low sensitivity. Dynamic processes such as conformational changes or binding events can induce drastic effects on NMR spectra in response to variations in chemical exchange (CE) rate, which can lead to new strategies in the design of supramolecular sensors through the control and monitoring of CE rate. Here, we present an indirect NMR anion sensing technique in which increased CE rate, due to anion-induced conformational flexibility of a relatively rigid structure of a novel sensor, allows ultrasensitive anion detection as low as 120 nM.

  4. ELISE NMR: Experimental liquid sealing of NMR samples

    Science.gov (United States)

    Wieruszeski, Jean-Michel; Landrieu, Isabelle; Hanoulle, Xavier; Lippens, Guy

    2006-08-01

    We present a simple, generally applicable approach to prevent sample evaporation when working at elevated temperatures in high resolution NMR. It consists of experimentally sealing the NMR sample by a second liquid (Experimental Liquid Sealing, ELISE). For aqueous samples, we identified the mineral oil commonly used in PCR application as the best candidate, because it contains only a very limited amount of water-soluble contaminants, is stable over time and heat resistant. The procedure does not interfere with shim settings, and is compatible with a wide variety of samples, including oligosaccharides and proteins. For chloroform samples, a simple drop of water allows to efficiently seal the sample, avoiding solvent evaporation even over lengthy time periods.

  5. Dynamical properties of confined supercooled water: an NMR study

    Science.gov (United States)

    Mallamace, Francesco; Broccio, Matteo; Corsaro, Carmelo; Faraone, Antonio; Liu, Li; Mou, Chung-Yuan; Chen, Sow-Hsin

    2006-09-01

    We report a set of dynamical data of confined water measured in a very deeply supercooled regime (290-190 K). Water is contained in silica matrices (MCM-41-S) which consist of 1D cylindrical pores with diameters d = 14,18 and 24 Å. When confined in these tubular pores, water does not crystallize, and can be supercooled well below 200 K. We use the NMR technique to obtain the characteristic proton relaxation time-constants (the spin-lattice relaxation time-constant T1 and the spin-spin relaxation time-constant T2) and a direct measurement of the self-diffusion coefficient in the whole temperature range. We give evidence of the existence of a fragile-to-strong dynamic crossover (FSC) at TL = 225 K from the temperature dependence of the self-diffusion coefficient. A combination of the NMR self-diffusion coefficient with the average translational relaxation time, as measured by quasi-elastic neutron scattering, shows a well defined decoupling of transport coefficients, i.e. the breakdown of the Stokes-Einstein relation, on approaching the crossover temperature TL.

  6. Dynamical properties of confined supercooled water: an NMR study

    Energy Technology Data Exchange (ETDEWEB)

    Mallamace, Francesco [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Broccio, Matteo [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Corsaro, Carmelo [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Faraone, Antonio [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Liu Li [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Mou, C-Y [Department of Chemistry, National Taiwan University, Taipei, Taiwan (China); Chen, S-H [Department of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2006-09-13

    We report a set of dynamical data of confined water measured in a very deeply supercooled regime (290-190 K). Water is contained in silica matrices (MCM-41-S) which consist of 1D cylindrical pores with diameters d = 14,18 and 24 A. When confined in these tubular pores, water does not crystallize, and can be supercooled well below 200 K. We use the NMR technique to obtain the characteristic proton relaxation time-constants (the spin-lattice relaxation time-constant T1 and the spin-spin relaxation time-constant T2) and a direct measurement of the self-diffusion coefficient in the whole temperature range. We give evidence of the existence of a fragile-to-strong dynamic crossover (FSC) at T{sub L} = 225 K from the temperature dependence of the self-diffusion coefficient. A combination of the NMR self-diffusion coefficient with the average translational relaxation time, as measured by quasi-elastic neutron scattering, shows a well defined decoupling of transport coefficients, i.e. the breakdown of the Stokes-Einstein relation, on approaching the crossover temperature T{sub L}.

  7. Assessment of DFT functionals with fluorine-fluorine coupling constants

    Science.gov (United States)

    García de la Vega, J. M.; San Fabián, J.

    2015-07-01

    Density functional theory (DFT) calculations of nuclear magnetic resonance (NMR) spin-spin coupling constants (SSCCs) provide an important contribution for understanding experimentally observed values. It is known that calculated SSCCs using DFT methods correlate well with those experimentally measured. Unlike most of SSCCs, in fluorine compounds, fluorine-fluorine SSCC JFF shows that the Fermi contact (FC) term is not dominant, particularly for JFF in polyfluorinated organic molecules. In order to devise a DFT approach that would correctly reproduce the variation of SSCCs within a series of fluorine compounds, we test several DFT-based approaches, using different exchange and correlation functionals. Isotropic contributions to NMR fluorine-fluorine coupling constants (FC, spin-dipolar, SD, paramagnetic spin-orbit, PSO, and diamagnetic spin-orbit, DSO) have been calculated. Results show that DFT methods give appropriate values for nJFF (n = 4 to 7), while for geminal and vicinal JFF present large deviations from experimental values. For the latter SSCCs (2JFF and 3JFF), the four contributions (FC, SD, PSO and DSO) are analysed as a function of the local and nonlocal exchange in 1,1- and 1,2-difluoroethylene. Although FC term is not dominant for these SSCCs, the variation of this contribution with exchange is remarkable. On the other hand, SD and PSO contributions can be suitably computed without and with exact exchange, respectively. This article is dedicated to the memory of Prof. N. C. Handy, whose contributions to the development of Theoretical Chemistry have been widely recognized.

  8. Nuclear magnetic resonance shielding constants in XH4 group XIV hydrides

    Science.gov (United States)

    Jaszuński, Michał; Ruud, Kenneth

    2006-07-01

    Self-consistent field and multiconfigurational self-consistent field wave functions are used to analyse NMR shielding constants in the XH4 hydrides, X = C, Si, Ge, Sn and Pb. All relativistic corrections to order α4, where α is the fine structure constant, are included in the evaluation of the perturbation corrections to the non-relativistic shielding constants. Each of the relativistic corrections is compared to the results obtained for group XVI and XVII hydrides and noble-gas atoms. For the heavy nuclei, the computed relativistic corrections can be used to improve the absolute shielding scale.

  9. NMR characteristics of intracellular K in the rat salivary gland: A sup 39 K NMR study using double-quantum filtering

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Yoshiteru; Murakami, Masataka; Suzuki, Eiji; Watari, Hiroshi (National Institute for Physiological Sciences, Myodaiji, Okazaki (Japan)); Nagayama, Kuniaki (JEOL ltd., Akishima, Tokyo (Japan))

    1990-01-23

    Intracellular K of the perfused rat mandibular salivary gland was measured by {sup 39}K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25{degree}C. The resonance due to intracellular K consisted of two Lorentzian signals. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 {times} 10 {sup {minus}8} s and a quadrupolar coupling constant ca. 1.4 MHz.

  10. Photochromism of indolino-benzopyrans studied by NMR and UV-visible spectroscopy

    Directory of Open Access Journals (Sweden)

    2006-01-01

    Full Text Available The synthesis of photochromic 3,3-di( 4 ′ -fluorophenyl-3H-benzopyrans fused to an indole moiety is described. The structures of photomerocyanines elucidated by NMR spectroscopy and spectrokinetic data ( λ max⁡ of colored form, colorability, and rate constant of bleaching obtained by UV-visible spectroscopy are reported.

  11. Photochromism of indolino-benzopyrans studied by NMR and UV-visible spectroscopy

    OpenAIRE

    S. Delbaere; J. Berthet; M. A. Salvador; Vermeersch, G.; Oliveira,M. M.

    2006-01-01

    The synthesis of photochromic 3,3-di(4′-fluorophenyl)-3H-benzopyrans fused to an indole moiety is described. The structures of photomerocyanines elucidated by NMR spectroscopy and spectrokinetic data (λmax⁡ of colored form, colorability, and rate constant of bleaching) obtained by UV-visible spectroscopy are reported.

  12. Self-Association of N-Methylacetamide Examined by Infrared and NMR Spectroscopies

    Science.gov (United States)

    Schenck, Heather L.; Hui, KaWai

    2011-01-01

    These spectroscopic experiments investigate polarity and concentration effects on self-association behavior in N-methylacetamide. Inquiry can be limited to the concentration dependence of hydrogen bonding and estimation of dimerization constant (NMR studies) or to the effect of solvent polarity on extent of hydrogen bonding (IR studies). The…

  13. Self-Association of N-Methylacetamide Examined by Infrared and NMR Spectroscopies

    Science.gov (United States)

    Schenck, Heather L.; Hui, KaWai

    2011-01-01

    These spectroscopic experiments investigate polarity and concentration effects on self-association behavior in N-methylacetamide. Inquiry can be limited to the concentration dependence of hydrogen bonding and estimation of dimerization constant (NMR studies) or to the effect of solvent polarity on extent of hydrogen bonding (IR studies). The…

  14. From the Rydberg constant to the fundamental constants metrology; De la constante de Rydberg a la metrologie des constantes fondamentales

    Energy Technology Data Exchange (ETDEWEB)

    Nez, F

    2005-06-15

    This document reviews the theoretical and experimental achievements of the author since the beginning of his scientific career. This document is dedicated to the spectroscopy of hydrogen, deuterium and helium atoms. The first part is divided into 6 sub-sections: 1) the principles of hydrogen spectroscopy, 2) the measurement of the 2S-nS/nD transitions, 3) other optical frequency measurements, 4) our contribution to the determination of the Rydberg constant, 5) our current experiment on the 1S-3S transition, 6) the spectroscopy of the muonic hydrogen. Our experiments have improved the accuracy of the Rydberg Constant by a factor 25 in 15 years and we have achieved the first absolute optical frequency measurement of a transition in hydrogen. The second part is dedicated to the measurement of the fine structure constant and the last part deals with helium spectroscopy and the search for optical references in the near infrared range. (A.C.)

  15. SOPPA and CCSD vibrational corrections to NMR indirect spin-spin coupling constants of small hydrocarbons

    DEFF Research Database (Denmark)

    Faber, Rasmus; Sauer, Stephan P. A.

    2015-01-01

    are in the order of 5 Hz for the one-bond carbon-hydrogen couplings and about 1 Hz or smaller for the other couplings apart from the one-bond carbon-carbon coupling (11 Hz) and the twobond carbon-hydrogen coupling (4 Hz) in ethyne. However, not for all couplings lead the inclusion of zero-point vibrational...

  16. Intercalation complex of proflavine with DNA: Structure and dynamics by solid-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Pei; Juang, Chilong; Harbison, G.S. (State Univ. of New York, Stony Brook (USA))

    1990-07-06

    The structure of the complex formed between the intercalating agent proflavine and fibrous native DNA was studied by one- and two-dimensional high-resolution solid-state nuclear magnetic resonance (NMR). Carbon-13-labeled proflavine was used to show that the drug is stacked with the aromatic ring plane perpendicular to the fiber axis and that it is essentially immobile. Natural abundance carbon-13 NMR of the DNA itself shows that proflavine binding does not change the puckering of the deoxyribose ring. However, phosphorus-31 NMR spectra show profound changes in the orientation of the phosphodiester grouping on proflavine binding, with some of the phosphodiesters tilting almost parallel to the helix axis, and a second set almost perpendicular. The first group to the phosphodiesters probably spans the intercalation sites, whereas the tilting of the second set likely compensates for the unwinding of the DNA by the intercalator.

  17. Interaction of bolaform surfactants with p-sulfonatocalix[4]arene: the role of two positive charges in the binding.

    Science.gov (United States)

    Francisco, Vitor; Garcia-Rio, Luis

    2014-06-17

    The inclusion binding manners of bolaform surfactants of type C(n)R6(2+) 2Br(-) with different spacer lengths (n = 6, 12) and terminal headgroup volumes (R = methyl, ethyl) by p-sulfonatocalix[4]arene were studied. The combination of ITC parameters (binding constants and complexation enthalpy and entropy) and NMR chemical shifts and NOE cross-peaks obtained upon complexation allows us to propose different binding modes. The results point out that the spacer length has an influence on the binding stoichiometry. The bolaforms with larger spacer lengths between polar head groups enable the formation of 2:1 complexes in addition to 1:1, while with the shorter spacer they form only 1:1 complexes. On the other hand, the formation of 1:1 complexes is not affected by the headgroup volume or the spacer length of the bolaform. Unexpectedly, a complex binding mode was observed where both positive charged terminal groups of the bolaform are accommodated in the cavity of the calixarene. The inclusion of both terminal groups of the guests can be related to its own structure but also evidence the high flexibility of the calixarene.

  18. Mirror QCD and Cosmological Constant

    CERN Document Server

    Pasechnik, Roman; Teryaev, Oleg

    2016-01-01

    An analog of Quantum Chromo Dynamics (QCD) sector known as mirror QCD (mQCD) can affect the cosmological evolution and help in resolving the Cosmological Constant problem. In this work, we explore an intriguing possibility for a compensation of the negative QCD vacuum contribution to the ground state energy density of the universe by means of a positive contribution from the chromomagnetic gluon condensate in mQCD. The trace anomaly compensation condition and the form of the mQCD coupling constant in the infrared limit have been proposed by analysing a partial non-perturbative solution of the Einstein--Yang-Mills equations of motion.

  19. Spatial Variations of Fundamental Constants

    CERN Document Server

    Barrow, John D; Barrow, John D.; Toole, Chris O'

    1999-01-01

    We show that observational limits on the possible time variation of constants of Nature are significantly affected by allowing for both space and time variation. Bekenstein's generalisation of Maxwell's equations to allow for cosmological variation of $alpha$ is investigated in a universe containing spherically symmetric inhomogeneities. The time variation of $alpha$ is determined by the local matter density and hence limits obtained in high-density geophysical enviroments are far more constraining than those obtained at high redshift. This new feature is expected to be a property of a wide class of theories for the variation of constants.

  20. Bouncing universes with varying constants

    Energy Technology Data Exchange (ETDEWEB)

    Barrow, John D [DAMTP, Centre for Mathematical Sciences, Cambridge University, Wilberforce Road, Cambridge CB3 0WA (United Kingdom); Kimberly, Dagny [Theoretical Physics, Blackett Laboratory, Imperial College, Prince Consort Road, London SW7 2BZ (United Kingdom); Magueijo, Joao [Theoretical Physics, Blackett Laboratory, Imperial College, Prince Consort Road, London SW7 2BZ (United Kingdom)

    2004-09-21

    We investigate the behaviour of exact closed bouncing Friedmann universes in theories with varying constants. We show that the simplest BSBM varying alpha theory leads to a bouncing universe. The value of alpha increases monotonically, remaining approximately constant during most of each cycle, but increasing significantly around each bounce. When dissipation is introduced we show that in each new cycle the universe expands for longer and to a larger size. We find a similar effect for closed bouncing universes in Brans-Dicke theory, where G also varies monotonically in time from cycle to cycle. Similar behaviour occurs also in varying speed of light theories.

  1. Bouncing Universes with Varying Constants

    CERN Document Server

    Barrow, J D; Magueijo, J; Barrow, John D.; Kimberly, Dagny; Magueijo, Joao

    2004-01-01

    We investigate the behaviour of exact closed bouncing Friedmann universes in theories with varying constants. We show that the simplest BSBM varying-alpha theory leads to a bouncing universe. The value of alpha increases monotonically, remaining approximately constant during most of each cycle, but increasing significantly around each bounce. When dissipation is introduced we show that in each new cycle the universe expands for longer and to a larger size. We find a similar effect for closed bouncing universes in Brans-Dicke theory, where $G$ also varies monotonically in time from cycle to cycle. Similar behaviour occurs also in varying speed of light theories.

  2. Gravitational Instantons and Cosmological Constant

    CERN Document Server

    Cyriac, Josily

    2015-01-01

    The cosmological dynamics of an otherwise empty universe in the presence of vacuum fields is considered. Quantum fluctuations at the Planck scale leads to a dynamical topology of space-time at very small length scales, which is dominated by compact gravitational instantons. The Planck scale vacuum energy acts as a source for the curvature of the these compact gravitational instantons and decouples from the large scale energy momentum tensor of the universe, thus making the observable cosmological constant vanish. However, a Euclidean functional integral over all possible topologies of the gravitational instantons generates a small non-zero value for the large scale cosmological constant, which agrees with the present observations.

  3. NMR analysis of the architecture and functional remodeling of a modular multidomain protein, RPA.

    Science.gov (United States)

    Brosey, Chris A; Chagot, Marie-Eve; Ehrhardt, Mark; Pretto, Dalyir I; Weiner, Brian E; Chazin, Walter J

    2009-05-13

    Modular proteins with multiple domains tethered by flexible linkers have variable global architectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation.

  4. Constant domain-regulated antibody catalysis.

    Science.gov (United States)

    Sapparapu, Gopal; Planque, Stephanie; Mitsuda, Yukie; McLean, Gary; Nishiyama, Yasuhiro; Paul, Sudhir

    2012-10-19

    Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

  5. The association constant of 5',8-cyclo-2'-deoxyguanosine with cytidine

    Directory of Open Access Journals (Sweden)

    Amedeo eCapobianco

    2015-03-01

    Full Text Available The association of 5',8-cyclo-2'-deoxyguanosine (cdG, a DNA tandem lesion, with its complementary base cytosine has been studied by voltammetry and NMR in chloroform, using properly silylated derivatives of the two nucleobases for increasing their solubilities. Both voltammetric data and NMR titrations indicated that the Watson-Crick complex of cytidine with cdG is weaker than that with guanosine, the difference being approximately of one order of magnitude between the two association constants.

  6. Binding Procurement

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  7. Determination of formation constants and structural characterization of cyclodextrin inclusion complexes with two phenolic isomers: carvacrol and thymol

    Directory of Open Access Journals (Sweden)

    Miriana Kfoury

    2016-01-01

    Full Text Available Carvacrol and thymol have been widely studied for their ability to control food spoilage and to extend shelf-life of food products due to their antimicrobial and antioxidant activities. However, they suffer from poor aqueous solubility and pronounced flavoring ability that limit their application in food systems. These drawbacks could be surpassed by encapsulation in cyclodextrins (CDs. Applications of their inclusion complexes with CDs were reported without investigating the inclusion phenomenon in deep. In this study, inclusion complexes were characterized in terms of formation constants (Kf, complexation efficiency (CE, CD:guest molar ratio and increase in bulk formulation by using an UV–visible competitive method, phase solubility studies as well as 1H and DOSY 1H NMR titration experiments. For the first time, a new algorithmic treatment that combines the chemical shifts and diffusion coefficients variations for all guest protons was applied to calculate Kf. The position of the hydroxy group in carvacrol and thymol did not affect the stoichiometry of the inclusion complexes but led to a different binding stability with CDs. 2D ROESY NMR experiments were also performed to prove the encapsulation and illustrate the stable 3D conformation of the inclusion complexes. The structural investigation was accomplished with molecular modeling studies. Finally, the radical scavenging activity of carvacrol and thymol was evaluated by the ABTS radical scavenging assay. An improvement of this activity was observed upon encapsulation. Taken together, these results evidence that the encapsulation in CDs could be valuable for applications of carvacrol and thymol in food.

  8. RECENT PROGRESS IN BIOMOLECULAR NMR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Structural genomics and proteomics were born from the understanding that functions of a protein are dictated by its 3D structure and dynamics. To understand protein functions on a genomic scale, we must know protein structures on a genomic scale. High resolution NMR can be used for this purpose. Traditional multidimensional NMR structure determination protocols become ineffective for structural genomics since to obtain a structure of a small protein of 15kD requires many months of painstaking spectral analysis and modeling. Recent advances in magnet and probe technology and in experimental methods have expanded the range of proteins amenable to structure determination and make the large scale structure determination possible. These advances are (1) effective expression systems for protein production, (2) introduction of cryoprobe, (3) structure determination with the use of the minimal amount of structural restraints obtained from the chemical shifts, residual dipolar couplings, NOEs, and computer modeling. In this talk,Iwill briefly outline these developments and related works done in our NMR lab.

  9. NMR-Based Milk Metabolomics

    Directory of Open Access Journals (Sweden)

    Hanne C. Bertram

    2013-04-01

    Full Text Available Milk is a key component in infant nutrition worldwide and, in the Western parts of the world, also in adult nutrition. Milk of bovine origin is both consumed fresh and processed into a variety of dairy products including cheese, fermented milk products, and infant formula. The nutritional quality and processing capabilities of bovine milk is closely associated to milk composition. Metabolomics is ideal in the study of the low-molecular-weight compounds in milk, and this review focuses on the recent nuclear magnetic resonance (NMR-based metabolomics trends in milk research, including applications linking the milk metabolite profiling with nutritional aspects, and applications which aim to link the milk metabolite profile to various technological qualities of milk. The metabolite profiling studies encompass the identification of novel metabolites, which potentially can be used as biomarkers or as bioactive compounds. Furthermore, metabolomics applications elucidating how the differential regulated genes affects milk composition are also reported. This review will highlight the recent advances in NMR-based metabolomics on milk, as well as give a brief summary of when NMR spectroscopy can be useful for gaining a better understanding of how milk composition is linked to nutritional or quality traits.

  10. Hyperpolarized 131Xe NMR spectroscopy

    Science.gov (United States)

    Stupic, Karl F.; Cleveland, Zackary I.; Pavlovskaya, Galina E.; Meersmann, Thomas

    2011-01-01

    Hyperpolarized (hp) 131Xe with up to 2.2% spin polarization (i.e., 5000-fold signal enhancement at 9.4 T) was obtained after separation from the rubidium vapor of the spin-exchange optical pumping (SEOP) process. The SEOP was applied for several minutes in a stopped-flow mode, and the fast, quadrupolar-driven T1 relaxation of this spin I = 3/2 noble gas isotope required a rapid subsequent rubidium removal and swift transfer into the high magnetic field region for NMR detection. Because of the xenon density dependent 131Xe quadrupolar relaxation in the gas phase, the SEOP polarization build-up exhibits an even more pronounced dependence on xenon partial pressure than that observed in 129Xe SEOP. 131Xe is the only stable noble gas isotope with a positive gyromagnetic ratio and shows therefore a different relative phase between hp signal and thermal signal compared to all other noble gases. The gas phase 131Xe NMR spectrum displays a surface and magnetic field dependent quadrupolar splitting that was found to have additional gas pressure and gas composition dependence. The splitting was reduced by the presence of water vapor that presumably influences xenon-surface interactions. The hp 131Xe spectrum shows differential line broadening, suggesting the presence of strong adsorption sites. Beyond hp 131Xe NMR spectroscopy studies, a general equation for the high temperature, thermal spin polarization, P, for spin I⩾1/2 nuclei is presented.

  11. Decay Constants of Vector Mesons

    Institute of Scientific and Technical Information of China (English)

    LI Heng-Mei; WAN Shao-Long

    2008-01-01

    @@ The light vector mesons are studied within the framework of the Bethe-Salpeter equation with the vector-vectortype flat-bottom potential The Bethe-Salpeter wavefunctions and the decay constants of the vector mesons are obtained. All the obtained results, fρ, fφ, and fΚ* , are in agreement with the experimental values, respectively.

  12. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

    2015-04-15

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

  13. The 1% concordance Hubble constant

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, C. L.; Larson, D.; Weiland, J. L. [Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 (United States); Hinshaw, G., E-mail: cbennett@jhu.edu [Department of Physics and Astronomy, University of British Columbia, Vancouver, BC V6T 1Z1 (Canada)

    2014-10-20

    The determination of the Hubble constant has been a central goal in observational astrophysics for nearly a hundred years. Extraordinary progress has occurred in recent years on two fronts: the cosmic distance ladder measurements at low redshift and cosmic microwave background (CMB) measurements at high redshift. The CMB is used to predict the current expansion rate through a best-fit cosmological model. Complementary progress has been made with baryon acoustic oscillation (BAO) measurements at relatively low redshifts. While BAO data do not independently determine a Hubble constant, they are important for constraints on possible solutions and checks on cosmic consistency. A precise determination of the Hubble constant is of great value, but it is more important to compare the high and low redshift measurements to test our cosmological model. Significant tension would suggest either uncertainties not accounted for in the experimental estimates or the discovery of new physics beyond the standard model of cosmology. In this paper we examine in detail the tension between the CMB, BAO, and cosmic distance ladder data sets. We find that these measurements are consistent within reasonable statistical expectations and we combine them to determine a best-fit Hubble constant of 69.6 ± 0.7 km s{sup –1} Mpc{sup –1}. This value is based upon WMAP9+SPT+ACT+6dFGS+BOSS/DR11+H {sub 0}/Riess; we explore alternate data combinations in the text. The combined data constrain the Hubble constant to 1%, with no compelling evidence for new physics.

  14. Variation of fundamental constants: theory

    Science.gov (United States)

    Flambaum, Victor

    2008-05-01

    Theories unifying gravity with other interactions suggest temporal and spatial variation of the fundamental ``constants'' in expanding Universe. There are some hints for the variation of different fundamental constants in quasar absorption spectra and Big Bang nucleosynthesis data. A large number of publications (including atomic clocks) report limits on the variations. We want to study the variation of the main dimensionless parameters of the Standard Model: 1. Fine structure constant alpha (combination of speed of light, electron charge and Plank constant). 2. Ratio of the strong interaction scale (LambdaQCD) to a fundamental mass like electron mass or quark mass which are proportional to Higgs vacuum expectation value. The proton mass is propotional to LambdaQCD, therefore, the proton-to-electron mass ratio comes into this second category. We performed necessary atomic, nuclear and QCD calculations needed to study variation of the fundamental constants using the Big Bang Nucleosynthsis, quasar spectra, Oklo natural nuclear reactor and atomic clock data. The relative effects of the variation may be enhanced in transitions between narrow close levels in atoms, molecules and nuclei. If one will study an enhanced effect, the relative value of systematic effects (which are not enhanced) may be much smaller. Note also that the absolute magnitude of the variation effects in nuclei (e.g. in very narrow 7 eV transition in 229Th) may be 5 orders of magnitude larger than in atoms. A different possibility of enhancement comes from the inversion transitions in molecules where splitting between the levels is due to the quantum tunneling amplitude which has strong, exponential dependence on the electron to proton mass ratio. Our study of NH3 quasar spectra has already given the best limit on the variation of electron to proton mass ratio.

  15. Varying Fine-Structure Constant and the Cosmological Constant Problem

    CERN Document Server

    Fujii, Y

    2003-01-01

    We start with a brief account of the latest analysis of the Oklo phenomenon providing the still most stringent constraint on time-variability of the fine- structure constant $\\alpha$. Comparing this with the recent result from the measurement of distant QSO's appears to indicate a non-uniform time-dependence, which we argue to be related to another recent finding of the accelerating universe. This view is implemented in terms of the scalar-tensor theory, applied specifically to the small but nonzero cosmological constant. Our detailed calculation shows that these two phenomena can be understood in terms of a common origin, a particular behavior of the scalar field, dilaton. We also sketch how this theoretical approach makes it appropriate to revisit non- Newtonian gravity featuring small violation of Weak Equivalence Principle at medium distances.

  16. Varying Fine-Structure Constant and the Cosmological Constant Problem

    Science.gov (United States)

    Fujii, Yasunori

    We start with a brief account of the latest analysis of the Oklo phenomenon providing the still most stringent constraint on time variability of the fine-structure constant α. Comparing this with the recent result from the measurement of distant QSO's appears to indicate a non-uniform time-dependence, which we argue to be related to another recent finding of the accelerating universe. This view is implemented in terms of the scalar-tensor theory, applied specifically to the small but nonzero cosmological constant. Our detailed calculation shows that these two phenomena can be understood in terms of a common origin, a particular behavior of the scalar field, dilaton. We also sketch how this theoretical approach makes it appropriate to revisit non-Newtonian gravity featuring small violation of Weak Equivalence Principle at medium distances.

  17. New Quasar Studies Keep Fundamental Physical Constant Constant

    Science.gov (United States)

    2004-03-01

    Very Large Telescope sets stringent limit on possible variation of the fine-structure constant over cosmological time Summary Detecting or constraining the possible time variations of fundamental physical constants is an important step toward a complete understanding of basic physics and hence the world in which we live. A step in which astrophysics proves most useful. Previous astronomical measurements of the fine structure constant - the dimensionless number that determines the strength of interactions between charged particles and electromagnetic fields - suggested that this particular constant is increasing very slightly with time. If confirmed, this would have very profound implications for our understanding of fundamental physics. New studies, conducted using the UVES spectrograph on Kueyen, one of the 8.2-m telescopes of ESO's Very Large Telescope array at Paranal (Chile), secured new data with unprecedented quality. These data, combined with a very careful analysis, have provided the strongest astronomical constraints to date on the possible variation of the fine structure constant. They show that, contrary to previous claims, no evidence exist for assuming a time variation of this fundamental constant. PR Photo 07/04: Relative Changes with Redshift of the Fine Structure Constant (VLT/UVES) A fine constant To explain the Universe and to represent it mathematically, scientists rely on so-called fundamental constants or fixed numbers. The fundamental laws of physics, as we presently understand them, depend on about 25 such constants. Well-known examples are the gravitational constant, which defines the strength of the force acting between two bodies, such as the Earth and the Moon, and the speed of light. One of these constants is the so-called "fine structure constant", alpha = 1/137.03599958, a combination of electrical charge of the electron, the Planck constant and the speed of light. The fine structure constant describes how electromagnetic forces hold

  18. The Doppler effect in NMR spectroscopy.

    Science.gov (United States)

    Guéron, Maurice

    2003-02-01

    An NMR sample may be subject to motions, such as those due to sample spinning or to liquid flow. Is the spectrum of such a sample affected by the Doppler effect? The question arises because, instrumental dimensions being much shorter than the wavelength, it is the near-field of the precessing magnetic moment which couples to the receiver coil, rather than the radiated far-field. We expand the near-field into plane propagating waves. For each such wave there is another one with the same amplitude, propagating in the opposite direction. The Doppler shifts are therefore equal and opposite. In the model case of a small fluid sample moving with constant velocity, this leads to a distribution of Doppler shifts which is symmetrical with respect to the unshifted frequency: there is no net spectral shift. We examine the possibility of observing the Doppler distribution in this case. We also consider the case of thermal motion of a gas. We draw attention to the resolved Doppler splitting of molecular rotational transitions in a supersonic burst as observed in a microwave resonator. We also mention briefly the Doppler effect in molecular beam spectroscopy.

  19. Constant-bandwidth constant-temperature hot-wire anemometer.

    Science.gov (United States)

    Ligeza, P

    2007-07-01

    A constant-temperature anemometer (CTA) enables the measurement of fast-changing velocity fluctuations. In the classical solution of CTA, the transmission band is a function of flow velocity. This is a minor drawback when the mean flow velocity does not significantly change, though it might lead to dynamic errors when flow velocity varies over a considerable range. A modification is outlined, whereby an adaptive controller is incorporated in the CTA system such that the anemometer's transmission band remains constant in the function of flow velocity. For that purpose, a second feedback loop is provided, and the output signal from the anemometer will regulate the controller's parameters such that the transmission bandwidth remains constant. The mathematical model of a CTA that has been developed and model testing data allow a through evaluation of the proposed solution. A modified anemometer can be used in measurements of high-frequency variable flows in a wide range of velocities. The proposed modification allows the minimization of dynamic measurement errors.

  20. 113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

    DEFF Research Database (Denmark)

    Kofod, Pauli; Bauer, Rogert; Danielsen, Eva

    1991-01-01

    113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper,zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits...... an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase....

  1. A NMR reverse diffusion filter for the simplification of spectra of complex mixtures and the study of drug receptor interactions.

    Science.gov (United States)

    Vega-Vázquez, M; Cobas, J C; Oliveira de Sousa, F F; Martin-Pastor, M

    2011-08-01

    A reverse diffusion filter NMR experiment (Drev) is proposed for the study of small molecules in binding with macromolecules. The filtering efficiency of Drev to eliminate the signals of the macromolecule is shown to be superior to conventional transverse relaxation filters at least for macromolecules containing a significant fraction of flexible residues. The Drev filter was also a useful complement for ligand-based NMR screening in combination with saturation transfer difference experiments.

  2. Prediction of recrystallization behavior of troglitazone/polyvinylpyrrolidone solid dispersion by solid-state NMR.

    Science.gov (United States)

    Ito, Atsutoshi; Watanabe, Tomoyuki; Yada, Shuichi; Hamaura, Takeshi; Nakagami, Hiroaki; Higashi, Kenjirou; Moribe, Kunikazu; Yamamoto, Keiji

    2010-01-04

    The purpose of this study was to elaborate the relationship between the (13)C CP/MAS NMR spectra and the recrystallization behavior during the storage of troglitazone solid dispersions. The solid dispersions were prepared by either the solvent method or by co-grinding. The recrystallization behavior under storage conditions at 40 degrees C/94% RH was evaluated by the Kolmogorov-Johnson-Mehl-Avrami (KJMA) equation. Solid dispersions prepared by the solvent method or by prolonged grinding brought about inhibition of the nucleation and the nuclei growth at the same time. No differences in the PXRD profiles were found in the samples prepared by the co-grinding and solvent methods, however, (13)C CP/MAS NMR showed significant differences in the spectra. The correlation coefficients using partial least square regression analysis between the PXRD profiles and the apparent nuclei-growth constant or induction period to nucleation were 0.1305 or 0.6350, respectively. In contrast, those between the (13)C CP/MAS NMR spectra and the constant or the period were 0.9916 or 0.9838, respectively. The (13)C CP/MAS NMR spectra had good correlation with the recrystallization kinetic parameters evaluated by the KJMA equation. Consequently, solid-state NMR was judged to be a useful tool for the prediction of the recrystallization behavior of solid dispersions.

  3. Three pion nucleon coupling constants

    CERN Document Server

    Arriola, E Ruiz; Perez, R Navarro

    2016-01-01

    There exist four pion nucleon coupling constants, $f_{\\pi^0, pp}$, $-f_{\\pi^0, nn}$, $f_{\\pi^+, pn} /\\sqrt{2}$ and $ f_{\\pi^-, np} /\\sqrt{2}$ which coincide when up and down quark masses are identical and the electron charge is zero. While there is no reason why the pion-nucleon-nucleon coupling constants should be identical in the real world, one expects that the small differences might be pinned down from a sufficiently large number of independent and mutually consistent data. Our discussion provides a rationale for our recent determination $$f_p^2 = 0.0759(4) \\, , \\quad f_{0}^2 = 0.079(1) \\,, \\quad f_{c}^2 = 0.0763(6) \\, , $$ based on a partial wave analysis of the $3\\sigma$ self-consistent nucleon-nucleon Granada-2013 database comprising 6713 published data in the period 1950-2013.

  4. Time-Varying Fundamental Constants

    Science.gov (United States)

    Olive, Keith

    2003-04-01

    Recent data from quasar absorption systems can be interpreted as arising from a time variation in the fine-structure constant. However, there are numerous cosmological, astro-physical, and terrestrial bounds on any such variation. These includes bounds from Big Bang Nucleosynthesis (from the ^4He abundance), the Oklo reactor (from the resonant neutron capture cross-section of Sm), and from meteoretic lifetimes of heavy radioactive isotopes. The bounds on the variation of the fine-structure constant are significantly strengthened in models where all gauge and Yukawa couplings vary in a dependent manner, as would be expected in unified theories. Models which are consistent with all data are severly challenged when Equivalence Principle constraints are imposed.

  5. Calculations of NMR properties for sI and sII clathrate hydrates of methane, ethane and propane.

    Science.gov (United States)

    Siuda, Paweł; Sadlej, Joanna

    2014-12-01

    Calculations of NMR parameters (the absolute shielding constants and the spin-spin coupling constants) for 5(12), 5(12)6(2) and 5(12)6(4) cages enclathrating CH4, C2H6 and C3H8 molecules are presented. The DFT/B3LYP/HuzIII-su3 level of theory was employed. The (13)C shielding constants of guest molecules are close to available experimental data. In two cases (the ethane in 5(12) and the propane in 5(12)6(2) cages) the (13)C shielding constants are reported for the first time. Inversion of the methyl/methylene (13)C and (1)H shielding constants order is found for propane in the 5(12)6(2) cage. Topological criteria are used to interpret the changes of values of NMR parameters of water molecules and they establish a connection between single cages and bulk crystal.

  6. Dynamic pulsed-field-gradient NMR

    CERN Document Server

    Sørland, Geir Humborstad

    2014-01-01

    Dealing with the basics, theory and applications of dynamic pulsed-field-gradient NMR NMR (PFG NMR), this book describes the essential theory behind diffusion in heterogeneous media that can be combined with NMR measurements to extract important information of the system being investigated. This information could be the surface to volume ratio, droplet size distribution in emulsions, brine profiles, fat content in food stuff, permeability/connectivity in porous materials and medical applications currently being developed. Besides theory and applications it will provide the readers with background knowledge on the experimental set-ups, and most important, deal with the pitfalls that are numerously present in work with PFG-NMR. How to analyze the NMR data and some important basic knowledge on the hardware will be explained, too.

  7. Theory of mirrored time domain sampling for NMR spectroscopy.

    Science.gov (United States)

    Ghosh, Arindam; Wu, Yibing; He, Yunfen; Szyperski, Thomas

    2011-12-01

    A generalized theory is presented for novel mirrored hypercomplex time domain sampling (MHS) of NMR spectra. It is the salient new feature of MHS that two interferograms are acquired with different directionality of time evolution, that is, one is sampled forward from time t=0 to the maximal evolution time tmax, while the second is sampled backward from t=0 to -tmax. The sampling can be accomplished in a (semi) constant time or non constant-time manner. Subsequently, the two interferograms are linearly combined to yield a complex time domain signal. The manifold of MHS schemes considered here is defined by arbitrary settings of sampling phases ('primary phase shifts') and amplitudes of the two interferograms. It is shown that, for any two given primary phase shifts, the addition theorems of trigonometric functions yield the unique linear combination required to form the complex signal. In the framework of clean absorption mode (CAM) acquisition of NMR spectra being devoid of residual dispersive signal components, 'secondary phase shifts' represent time domain phase errors which are to be eliminated. In contrast, such secondary phase shifts may be introduced by experimental design in order to encode additional NMR parameters, a new class of NMR experiments proposed here. For generalization, it is further considered that secondary phase shifts may depend on primary phase shifts and/or sampling directionality. In order to compare with MHS theory, a correspondingly generalized theory is derived for widely used hypercomplex ('States') sampling (HS). With generalized theory it is shown, first, that previously introduced 'canonical' schemes, characterized by primary phases being multiples of π/4, afford maximal intensity of the desired absorptive signals in the absence of secondary phase shifts, and second, how primary phases can be adjusted to maximize the signal intensity provided that the secondary phase shifts are known. Third, it is demonstrated that theory enables

  8. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Mavoungou, Chrystelle [Max Planck Institute for Biochemistry (Germany); Israel, Lars [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz [Max Planck Institute for Biochemistry (Germany); Noegel, Angelika A. [University of Cologne, Institute for Biochemistry (Germany); Schleicher, Michael [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Holak, Tad A. [Max Planck Institute for Biochemistry (Germany)

    2004-05-15

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state {sup 1}H-{sup 15}N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an {alpha}-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by {beta}-strands.

  9. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    Science.gov (United States)

    Mavoungou, Chrystelle; Israel, Lars; Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.

  10. Applications of NMR in Dairy Research

    Directory of Open Access Journals (Sweden)

    Anthony D. Maher

    2014-03-01

    Full Text Available NMR is a robust analytical technique that has been employed to investigate the properties of many substances of agricultural relevance. NMR was first used to investigate the properties of milk in the 1950s and has since been employed in a wide range of studies; including properties analysis of specific milk proteins to metabolomics techniques used to monitor the health of dairy cows. In this brief review, we highlight the different uses of NMR in the dairy industry.

  11. Studies on the structure of the complex of the boron neutron capture therapy drug, L-p-boronophenylalanine, with fructose and related carbohydrates: chemical and 13C NMR evidence for the beta-D-fructofuranose 2,3,6-(p-phenylalanylorthoboronate) structure.

    Science.gov (United States)

    Shull, B K; Spielvogel, D E; Head, G; Gopalaswamy, R; Sankar, S; Devito, K

    2000-02-01

    The complex of L-L-boronophenylalanine (L-p-BPA) with fructose has been used for the past 5 years in clinical trials of boron neutron capture therapy to treat both melanoma and glioblastoma multiforme. However, the structure of this complex in water buffered at physiologic pH has not been established. In the (1)H NMR spectra (D(2)O buffered at pD 7.4) of the complex of L-p-BPA with various carbohydrates, the upfield chemical shifts of the aromatic protons of L-p-BPA confirm that the boron atom is negatively charged and tetrahedral. In the (13)C NMR spectrum of the complex of L-p-BPA with U-(13)C labeled fructose, the chemical shifts and (1)J(CC) coupling constants are consistent with fructose adopting the beta-D-fructofuranose form. In addition, the (1)J(CC) coupling constants along with the binding constants measured for L-p-BPA with a series of monosaccharides and disaccharides seem to suggest that the beta-D-fructofuranose 2,3,6-(p-phenylalanylorthoboronate) structure strongly predominates, with free L-p-BPA and fructose the only other species detected. Copyright 2000 Wiley-Liss, Inc.

  12. Why isn't the solar constant a constant?

    CERN Document Server

    Li, K J; Xu, J C; Gao, P X; Yang, L H; Liang, H F; Zhan, L S

    2012-01-01

    In order to probe the mechanism of variations of the Solar Constant on the inter-solar-cycle scale, total solar irradiance (TSI, the so-called Solar Constant) in the time interval of 7 November 1978 to 20 September 2010 is decomposed into three components through the empirical mode decomposition and time-frequency analyses. The first component is the rotation signal, counting up to 42.31% of the total variation of TSI, which is understood to be mainly caused by large magnetic structures, including sunspot groups. The second is an annual-variation signal, counting up to 15.17% of the total variation, the origin of which is not known at this point in time. Finally, the third is the inter-solar-cycle signal, counting up to 42.52%, which are inferred to be caused by the network magnetic elements in quiet regions, whose magnetic flux ranges from $(4.27-38.01)\\times10^{19}$ Mx.

  13. Fine-structure constant: Is it really a constant?

    Science.gov (United States)

    Bekenstein, Jacob D.

    1982-03-01

    It is often claimed that the fine-structure "constant" α is shown to be strictly constant in time by a variety of astronomical and geophysical results. These constrain its fractional rate of change α˙α to at least some orders of magnitude below the Hubble rate H0. We argue that the conclusion is not as straightforward as claimed since there are good physical reasons to expect α˙α<

  14. Fourier Analysis and Structure Determination. Part II: Pulse NMR and NMR Imaging.

    Science.gov (United States)

    Chesick, John P.

    1989-01-01

    Uses simple pulse NMR experiments to discuss Fourier transforms. Studies the generation of spin echoes used in the imaging procedure. Shows that pulse NMR experiments give signals that are additions of sinusoids of differing amplitudes, frequencies, and phases. (MVL)

  15. Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy.

    Science.gov (United States)

    Mandala, Venkata S; Liao, Shu-Yu; Kwon, Byungsu; Hong, Mei

    2017-07-07

    The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus. (15)N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. NMR studies of the conformation of the natural sweetener rebaudioside A.

    Science.gov (United States)

    Steinmetz, Wayne E; Lin, Alvin

    2009-12-14

    Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four beta-D-glucopyranose units are attached to the aglycone steviol. Its (1)H and (13)C NMR spectra in pyridine-d(5) were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the beta-D-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.

  17. Generalised 2D-correlation NMR analysis of a wine fermentation.

    Science.gov (United States)

    Kirwan, Gemma M; Clark, Shona; Barnett, Neil W; Niere, Julie O; Adams, Michael J

    2008-11-23

    A wine fermentation has been monitored on a daily basis by (1)H NMR spectroscopy. Following data pre-processing that includes synthesis of the spectra to ensure all peaks are of constant half-width, the series of spectra were examined using generalised two-dimensional correlation techniques. Synchronous and asynchronous data maps have been generated and employed to interpret the changes in the fermentation process as a function of time. The results illustrate the potential of high resolution NMR with multivariate data analysis as a tool for process monitoring and the manner in which two-dimensional correlation mapping can aid in data interpretation.

  18. Synthesis and proton NMR spectroscopy of intra-vesicular gamma-aminobutyric acid (GABA).

    Science.gov (United States)

    Wang, Luke Y-J; Tong, Rong; Kohane, Daniel S

    2013-01-01

    We report the synthesis of vesicles containing gamma-aminobutyric acid (GABA), and their proton nuclear magnetic resonance ((1)H NMR) spectra. These vesicles were constructed to more closely mimic the intracellular environment wherein GABA exists. For this study, these GABA-containing vesicles were examined under (1)H NMR as a potential platform for future studies on the differences between aqueous phantoms, ex vivo brain extracts, and in vivo magnetic resonance spectroscopy results. We found that intra-vesicular GABA faithfully yielded the chemical shifts and J-coupling constants of free aqueous GABA, alongside the chemical shift signals of the vesicle wall.

  19. {sup 11}B-NMR spectroscopic study on the interaction of epinephrine and p-BPA

    Energy Technology Data Exchange (ETDEWEB)

    Ichihara, K.; Yoshino, K. [Shinshu Univ., Department of Chemistry, Matsumoto, Nagano (Japan)

    2000-10-01

    It is studied that p-BPA (p-bronophenylalanine) which formed complex with catechol functional group has interaction with epinephrine by {sup 11}B-NMR. Two {sup 11}B-NMR resonance signals were observed at pH 7.0. The signal at 29.6 ppm is assigned to p-BPA and at 10.8 ppm is assigned to that of complex. We can determine complex formation constants (logK') in various pH. (author)

  20. NMR studies of isotopically labeled RNA

    Energy Technology Data Exchange (ETDEWEB)

    Pardi, A. [Univ. of Colorado, Boulder, CO (United States)

    1994-12-01

    In summary, the ability to generate NMR quantities of {sup 15}N and {sup 13}C-labeled RNAs has led to the development of heteronuclear multi-dimensional NMR techniques for simplifying the resonance assignment and structure determination of RNAs. These methods for synthesizing isotopically labeled RNAs are only several years old, and thus there are still relatively few applications of heteronuclear multi-dimensional NMR techniques to RNA. However, given the critical role that RNAs play in cellular function, one can expect to see an increasing number of NMR structural studies of biologically active RNAs.