Science.gov

Sample records for nmr binding constant

  1. {sup 13}C and {sup 17}O NMR binding constant studies of uranyl carbonate complexes in near-neutral aqueous solution. Yucca Mountain Project Milestone Report 3351

    SciTech Connect

    Clark, D.L.; Newton, T.W.; Palmer, P.D.; Zwick, B.D.

    1995-01-01

    Valuable structural information, much of it unavailable by other methods, can be obtained about complexes in solution through NMR spectroscopy. From chemical shift and intensity measurements of complexed species, NMR can serve as a species-specific structural probe for molecules in solution and can be used to validate thermodynamic constants used in geochemical modeling. Fourier-transform nuclear magnetic resonance (FT-NMR) spectroscopy has been employed to study the speciation of uranium(VI) ions in aqueous carbonate solutions as a function of pH, ionic strength, carbonate concentration, uranium concentration, and temperature. Carbon-13 and oxygen-17 NMR spectroscopy were used to monitor the fractions, and hence thermodynamic binding constants of two different uranyl species U0{sub 2}(CO{sub 3}){sub 3}{sup 4{minus}} and (UO{sub 2}){sub 3}(CO{sub 3}){sub 6}{sup 6{minus}} in aqueous solution. Synthetic buffer solutions were prepared under the ionic strength conditions used in the NMR studies in order to obtain an accurate measure of the hydrogen ion concentration, and a discussion of pH = {minus}log(a{sub H}{sup +}) versus p[H] = {minus}log[H+] is provided. It is shown that for quantitative studies, the quantity p[H] needs to be used. Fourteen uranium(VI) binding constants recommended by the OECD NEA literature review were corrected to the ionic strengths employed in the NMR study using specific ion interaction theory (SIT), and the predicted species distributions were compared with the actual species observed by multinuclear NMR. Agreement between observed and predicted stability fields is excellent. This establishes the utility of multinuclear NMR as a species-specific tool for the study of the actinide carbonate complexation constants, and serves as a means for validating the recommendations provided by the OECD NEA.

  2. CONSTEQ - A program for association constants determination using solution NMR data

    NASA Astrophysics Data System (ADS)

    Floare, Cǎlin G.; Bogdan, Mircea

    2013-11-01

    CONSTEQ is a program developed in our group to determine association constants from NMR titration experiments using a non-linear regression algorithm. We use it essentially to determine binding constants of supramolecular inclusion compounds of cyclodextrins, at 1:1 stoichiometry. The implemented algorithm performs a global analysis, simultaneously fitting multiple experimental datasets. In this contribution we present its capabilities, its performance and all its latest developments.

  3. NMR Study of Strontium Binding by a Micaceous Mineral

    SciTech Connect

    Bowers, Geoffrey M.; Ravella, Ramesh; Komarneni, S.; Mueller, Karl T.

    2006-04-13

    The nature of strontium binding by soil minerals directly affects the transport and sequestration/remediation of radioactive strontium species released from leaking high-level nuclear waste storage tanks. However, the molecular-level structure of strontium binding sites has seldom been explored in phyllosilicate minerals by direct spectroscopic means and is not well-understood. In this work, we use solid-state NMR to analyze strontium directly and indirectly in a fully strontium-exchanged synthetic mica of nominal composition Na4Mg6Al4Si4O20F4. Thermogravimetric analysis, X-ray diffraction analysis, and NMR evidence supports that heat treatment at 500 °C for 4 h fully dehydrates the mica, creating a hydrogen-free interlayer. Analysis of the strontium NMR spectrum of the heat-treated mica shows a single strontium environment with a quadrupolar coupling constant of 9.02 MHz and a quadrupolar asymmetry parameter of 1.0. These quadrupolar parameters are consistent with a highly distorted and asymmetric coordination environment that would be produced by strontium cations without water in the coordination sphere bound deep within the ditrigonal holes. Evidence for at least one additional strontium environment, where proton-strontium couplings may occur, was found via a 1H-87Sr transfer of populations by double resonance NMR experiment. We conclude that the strontium cations in the proton-free interlayer are observable by 87Sr NMR and bound through electrostatic interactions as nine coordinate inner-sphere complexes sitting in the ditrigonal holes. Partially hydrated strontium cations invisible to direct 87Sr NMR are also present and located on the external mica surfaces, which are known to hydrate upon exposure to atmospheric moisture. These results demonstrate that modern pulsed NMR techniques and high fields can be used effectively to provide structural details of strontium binding by phyllosilicate minerals.

  4. NMR study of strontium binding by a micaceous mineral.

    PubMed

    Bowers, Geoffrey M; Ravella, Ramesh; Komarneni, Sridhar; Mueller, Karl T

    2006-04-13

    The nature of strontium binding by soil minerals directly affects the transport and sequestration/remediation of radioactive strontium species released from leaking high-level nuclear waste storage tanks. However, the molecular-level structure of strontium binding sites has seldom been explored in phyllosilicate minerals by direct spectroscopic means and is not well-understood. In this work, we use solid-state NMR to analyze strontium directly and indirectly in a fully strontium-exchanged synthetic mica of nominal composition Na(4)Mg(6)Al(4)Si(4)O(20)F(4). Thermogravimetric analysis, X-ray diffraction analysis, and NMR evidence supports that heat treatment at 500 degrees C for 4 h fully dehydrates the mica, creating a hydrogen-free interlayer. Analysis of the strontium NMR spectrum of the heat-treated mica shows a single strontium environment with a quadrupolar coupling constant of 9.02 MHz and a quadrupolar asymmetry parameter of 1.0. These quadrupolar parameters are consistent with a highly distorted and asymmetric coordination environment that would be produced by strontium cations without water in the coordination sphere bound deep within the ditrigonal holes. Evidence for at least one additional strontium environment, where proton-strontium couplings may occur, was found via a (1)H-(87)Sr transfer of populations by double resonance NMR experiment. We conclude that the strontium cations in the proton-free interlayer are observable by (87)Sr NMR and bound through electrostatic interactions as nine coordinate inner-sphere complexes sitting in the ditrigonal holes. Partially hydrated strontium cations invisible to direct (87)Sr NMR are also present and located on the external mica surfaces, which are known to hydrate upon exposure to atmospheric moisture. These results demonstrate that modern pulsed NMR techniques and high fields can be used effectively to provide structural details of strontium binding by phyllosilicate minerals. PMID:16599480

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Spin-rotation and NMR shielding constants in HCl

    SciTech Connect

    Jaszuński, Michał; Repisky, Michal; Demissie, Taye B.; Komorovsky, Stanislav; Malkin, Elena; Ruud, Kenneth; Garbacz, Piotr; Jackowski, Karol; Makulski, Włodzimierz

    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.

  7. Protein Dielectric Constants Determined from NMR Chemical Shift Perturbations

    PubMed Central

    Kukic, Predrag; Farrell, Damien; McIntosh, Lawrence P.; E., Bertrand García-Moreno; Jensen, Kristine Steen; Toleikis, Zigmantas; Teilum, Kaare; Nielsen, Jens Erik

    2015-01-01

    Understanding the connection between protein structure and function requires a quantitative understanding of electrostatic effects. Structure-based electrostatics calculations are essential for this purpose, but their use have been limited by a long-standing discussion on which value to use for the dielectric constants (εeff and εp) required in Coulombic models and Poisson-Boltzmann models. The currently used values for εeff and εp are essentially empirical parameters calibrated against thermodynamic properties that are indirect measurements of protein electric fields. We determine optimal values for εeff and εp by measuring protein electric fields in solution using direct detection of NMR chemical shift perturbations (CSPs). We measured CSPs in fourteen proteins to get a broad and general characterization of electric fields. Coulomb's law reproduces the measured CSPs optimally with a protein dielectric constant (εeff) from 3 to 13, with an optimal value across all proteins of 6.5. However, when the water-protein interface is treated with finite difference Poisson-Boltzmann calculations, the optimal protein dielectric constant (εp) rangedsfrom 2-5 with an optimum of 3. It is striking how similar this value is to the dielectric constant of 2-4 measured for protein powders, and how different it is from the εp of 6-20 used in models based on the Poisson-Boltzmann equation when calculating thermodynamic parameters. Because the value of εp = 3 is obtained by analysis of NMR chemical shift perturbations instead of thermodynamic parameters such as pKa values, it is likely to describe only the electric field and thus represent a more general, intrinsic, and transferable εp common to most folded proteins. PMID:24124752

  8. Binding of ionic species: a general approach to measuring binding constants and assessing affinities.

    PubMed

    Roelens, Stefano; Vacca, Alberto; Venturi, Chiara

    2009-03-01

    Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptor's binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown

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

    ERIC Educational Resources Information Center

    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)

  10. Radioligand Binding Assays for Determining Dissociation Constants of Phytohormone Receptors.

    PubMed

    Hellmuth, Antje; Calderón Villalobos, Luz Irina A

    2016-01-01

    In receptor-ligand interactions, dissociation constants provide a key parameter for characterizing binding. Here, we describe filter-based radioligand binding assays at equilibrium, either varying ligand concentrations up to receptor saturation or outcompeting ligand from its receptor with increasing concentrations of ligand analogue. Using the auxin coreceptor system, we illustrate how to use a saturation binding assay to determine the apparent dissociation constant (K D (') ) for the formation of a ternary TIR1-auxin-AUX/IAA complex. Also, we show how to determine the inhibitory constant (K i) for auxin binding by the coreceptor complex via a competition binding assay. These assays can be applied broadly to characterize a one-site binding reaction of a hormone to its receptor. PMID:27424743

  11. Analyzing and Interpreting NMR Spin-Spin Coupling Constants Using Molecular Orbital Calculations

    ERIC Educational Resources Information Center

    Autschbach, Jochen; Le Guennic, Boris

    2007-01-01

    Molecular orbital plots are used to analyze and interpret NMR spin-spin coupling constants, also known as J coupling constants. Students have accepted the concept of contributions to molecular properties from individual orbitals without the requirement to provide explicit equations.

  12. NMR structural analysis of Sleeping Beauty transposase binding to DNA

    PubMed Central

    E Carpentier, Claire; Schreifels, Jeffrey M; Aronovich, Elena L; Carlson, Daniel F; Hackett, Perry B; Nesmelova, Irina V

    2014-01-01

    The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA. PMID:24243759

  13. Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay.

    PubMed

    Romaniuk, Paul J

    2016-01-01

    Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the measurement of the effects of mutations in either the WT1 zinc finger domain or the putative binding sites within a promoter element or cellular RNA.

  14. Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR

    PubMed Central

    Gater, Deborah L.; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

    2014-01-01

    Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites. PMID:25418299

  15. Semi-empirical proton binding constants for natural organic matter

    NASA Astrophysics Data System (ADS)

    Matynia, Anthony; Lenoir, Thomas; Causse, Benjamin; Spadini, Lorenzo; Jacquet, Thierry; Manceau, Alain

    2010-03-01

    Average proton binding constants ( KH,i) for structure models of humic (HA) and fulvic (FA) acids were estimated semi-empirically by breaking down the macromolecules into reactive structural units (RSUs), and calculating KH,i values of the RSUs using linear free energy relationships (LFER) of Hammett. Predicted log KH,COOH and log KH,Ph-OH are 3.73 ± 0.13 and 9.83 ± 0.23 for HA, and 3.80 ± 0.20 and 9.87 ± 0.31 for FA. The predicted constants for phenolic-type sites (Ph-OH) are generally higher than those derived from potentiometric titrations, but the difference may not be significant in view of the considerable uncertainty of the acidity constants determined from acid-base measurements at high pH. The predicted constants for carboxylic-type sites agree well with titration data analyzed with Model VI (4.10 ± 0.16 for HA, 3.20 ± 0.13 for FA; Tipping, 1998), the Impermeable Sphere model (3.50-4.50 for HA; Avena et al., 1999), and the Stockholm Humic Model (4.10 ± 0.20 for HA, 3.50 ± 0.40 for FA; Gustafsson, 2001), but differ by about one log unit from those obtained by Milne et al. (2001) with the NICA-Donnan model (3.09 ± 0.51 for HA, 2.65 ± 0.43 for FA), and used to derive recommended generic values. To clarify this ambiguity, 10 high-quality titration data from Milne et al. (2001) were re-analyzed with the new predicted equilibrium constants. The data are described equally well with the previous and new sets of values ( R2 ⩾ 0.98), not necessarily because the NICA-Donnan model is overparametrized, but because titration lacks the sensitivity needed to quantify the full binding properties of humic substances. Correlations between NICA-Donnan parameters are discussed, but general progress is impeded by the unknown number of independent parameters that can be varied during regression of a model fit to titration data. The high consistency between predicted and experimental KH,COOH values, excluding those of Milne et al. (2001), gives faith in the proposed

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

    SciTech Connect

    Boonsri, Pornthip; Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Sem, Daniel S.

    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.

  17. Quantitative Analysis of Metabolic Mixtures by 2D 13C-Constant-Time TOCSY NMR Spectroscopy

    PubMed Central

    Bingol, Kerem; Zhang, Fengli; Bruschweiler-Li, Lei; Brüschweiler, Rafael

    2013-01-01

    An increasing number of organisms can be fully 13C-labeled, which has the advantage that their metabolomes can be studied by high-resolution 2D NMR 13C–13C constant-time (CT) TOCSY experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. Determination of quantitative metabolite concentrations is another key task. Because significant peak overlaps in 1D NMR spectra prevents straightforward quantification through 1D peak integrals, we demonstrate here the direct use of 13C–13C CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum-mechanical treatment of the TOCSY magnetization transfer at short and long mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino-acid mixtures. PMID:23773204

  18. Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets.

    PubMed

    Chen, Wan-Na; Nitsche, Christoph; Pilla, Kala Bharath; Graham, Bim; Huber, Thomas; Klein, Christian D; Otting, Gottfried

    2016-04-01

    Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group. PMID:26974502

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

    PubMed

    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. PMID:26670708

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

    PubMed

    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.

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

    PubMed

    Kutateladze, Andrei G; Mukhina, Olga A

    2014-09-01

    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.

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

    PubMed

    Kutateladze, Andrei G; Mukhina, Olga A

    2014-09-01

    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. PMID:25158224

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    SciTech Connect

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. )

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  5. NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases.

    PubMed

    Peti, Wolfgang; Page, Rebecca

    2016-01-01

    NMR spectroscopy and other solution methods are increasingly being used to obtain novel insights into the mechanisms by which MAPK regulatory proteins bind and direct the activity of MAPKs. Here, we describe how interactions between the MAPK p38α and its regulatory proteins are studied using NMR spectroscopy, isothermal titration calorimetry, and small angle X-ray scattering (SAXS). PMID:27514807

  6. Measurement of State-Specific Association Constants in Allosteric Sensors through Molecular Stapling and NMR.

    PubMed

    Moleschi, Kody J; Akimoto, Madoka; Melacini, Giuseppe

    2015-08-26

    Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e., state-selective molecular stapling through disulfide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA-RIα), for which the structures of the inactive and active states are available, as needed to design the state-selective disulfide bridges. Surprisingly, the PKA-RIα state-specific association constants are comparable to those of a structurally homologous domain with ∼10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.

  7. Investigations of acetaminophen binding to bovine serum albumin in the presence of fatty acid: Fluorescence and 1H NMR studies

    NASA Astrophysics Data System (ADS)

    Bojko, B.; Sułkowska, A.; Maciążek-Jurczyk, M.; Równicka, J.; Sułkowski, W. W.

    2009-04-01

    The binding of acetaminophen to bovine serum albumin (BSA) was studied by the quenching fluorescence method and the proton nuclear magnetic resonance technique ( 1H NMR). For fluorescence measurements 1-anilino-9-naphthalene sulfonate (ANS) hydrophobic probe was used to verify subdomain IIIA as acetaminophen's likely binding site. Three binding sites of acetaminophen in subdomain IIA of bovine serum albumin were found. Quenching constants calculated by the Stern-Volmer modified method were used to estimate the influence of myristic acid (MYR) on the drug binding to the albumin. The influence of [fatty acid]/[albumin] molar ratios on the affinity of the protein towards acetaminophen was described. Changes of chemical shifts and relaxation times of the drug indicated that the presence of MYR inhibits interaction in the AA-albumin complex. It is suggested that the elevated level of fatty acids does not significantly influence the pharmacokinetics of acetaminophen.

  8. Second order rate constants for intramolecular conversions: Application to gas-phase NMR relaxation times

    NASA Astrophysics Data System (ADS)

    Bauer, S. H.; Lazaar, K. I.

    1983-09-01

    The usually quoted expression for the second order rate constant, for a unimolecular reaction at the low pressure limit, is valid only for strictly irreversible processes. Its application to isomerization reactions (which are to some extent reversible) is demonstrably in error; corrected expressions have been published. Attention is directed to intramolecular conversions over low barriers, for which the inappropriateness of the unidirectional expression becomes obvious. For such isomerizations we propose a model which incorporates only operationally observable states, so that an essential conceptual ambiguity is avoided. Use of this model is illustrated for the syn⇄anti conversions of methyl nitrite, derived from a gas phase NMR coalescence curve (Mc:Tc). The present data suggest that during isomerization the alkyl nitrites may not be completely ergodic on a time scale of 10-9 s. A regional phase-space model is proposed which has the appropriate formalism to account for this behavior.

  9. O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities.

    PubMed

    Chen, Wan-Na; Kuppan, Kekini Vahini; Lee, Michael David; Jaudzems, Kristaps; Huber, Thomas; Otting, Gottfried

    2015-04-01

    O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional (1)H NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl (1)H NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant Kd (0.2 μM) of the complex between glutamate and the Escherichia coli aspartate/glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the (1)H NMR signal of the Tby tert-butyl group allows Kd measurements using less concentrated protein solutions than usual, providing access to Kd values 1 order of magnitude lower than established NMR methods that employ direct protein detection for Kd measurements. PMID:25789794

  10. NMR J-coupling constants in cisplatin derivatives studied by molecular dynamics and relativistic DFT.

    PubMed

    Sutter, Kiplangat; Truflandier, Lionel A; Autschbach, Jochen

    2011-06-01

    Solvent effects on J((195)Pt-(15)N) one-bond nuclear spin-spin coupling constants (J(PtN)) of cisplatin [cis-diamminedichloroplatinum(II)] and three cisplatin derivatives are investigated using a combination of density functional theory (DFT) based ab initio molecular dynamics (aiMD) and all-electron relativistic DFT NMR calculations employing the two-component relativistic zeroth-order regular approximation (ZORA). Good agreement with experiment is obtained when explicit solvent molecules are considered and when the computations are performed with a hybrid functional. Spin-orbit coupling causes only small effects on J(PtN) . Key factors contributing to the magnitude of coupling constants are elucidated, with the most significant being the presence of solvent as well as the quality of the density functional and basis set combination. The solvent effects are of the same magnitude as J(PtN) calculated for gas-phase geometries. However, the trends of J(PtN) among the complexes are already present in the gas phase. Results obtained with a continuum solvent model agree quite well with the aiMD results, provided that the Pt solvent-accessible radius is carefully chosen. The aiMD results support the existence of a partial hydrogen-bond-like inverse-hydration-type interaction affording a weak (1)J(Pt⋅⋅⋅H(w)) coupling between the complexes and the coordinating water molecule.

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

    PubMed

    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.

  12. Revisiting NMR through-space J(FF) spin-spin coupling constants for getting insight into proximate F---F interactions.

    PubMed

    Contreras, Rubén H; Llorente, Tomás; Ducati, Lucas Colucci; Tormena, Cláudio Francisco

    2014-07-10

    At present times it is usual practice to mark biological compounds replacing an H for an F atom to study, by means of (19)F NMR spectroscopy, aspects such as binding sites and molecular folding features. This interesting methodology could nicely be improved if it is known how proximity interactions on the F atom affect its electronic structure as gauged through high-resolution (19)F NMR spectroscopy. This is the main aim of the present work and, to this end, differently substituted peri-difluoronaphthalenes are chosen as model systems. In such compounds are rationalized some interesting aspects of the diamagnetic and paramagnetic parts of the (19)F nuclear magnetic shielding tensor as well as the transmission mechanisms for the PSO and FC contributions to (4)JF1F8 indirect nuclear spin-spin coupling constants.

  13. Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.

    PubMed

    Venkitakrishnan, Rani Parvathy; Benard, Outhiriaradjou; Max, Marianna; Markley, John L; Assadi-Porter, Fariba M

    2012-01-01

    Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.

  14. Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy.

    PubMed

    Rothenstein, Dirk; Claasen, Birgit; Omiecienski, Beatrice; Lammel, Patricia; Bill, Joachim

    2012-08-01

    Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach. PMID:22720657

  15. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    SciTech Connect

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji )

    1991-07-02

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with {sup 13}C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with (1-{sup 13}C)Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how {sup 13}C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

  16. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

    NASA Astrophysics Data System (ADS)

    Ding, Fei; Zhao, Guangyu; Chen, Shoucong; Liu, Feng; Sun, Ying; Zhang, Li

    2009-07-01

    The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants ( Ka) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M -1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be -3.634 kJ mol -1 and 72.66 J mol -1 K -1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Binding constants of phenylalanine for the four mononucleotides

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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

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

    SciTech Connect

    Schaefer, J.

    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.

  1. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  2. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

  3. The estimation of affinity constants for the binding of model peptides to DNA by equilibrium dialysis.

    PubMed Central

    Standke, K C; Brunnert, H

    1975-01-01

    The binding of lysine model peptides of the type Lys-X-Lys, Lys-X-X-Lys and Lys-X-X-X-Lys (X = different aliphatic and aromatic amino acids) has been studied by equilibrium dialysis. It was shown that the strong electrostatic binding forces generated by protonated amino groups of lysine can be distinguished from the weak forces stemming from neutral and aromatic spacer amino acids. The overall binding strength of the lysine model peptides is modified by these weak binding forces and the apparent binding constants are influenced more by the hydrophobic character of the spacer amino acid side chains than by the chainlength of the spacers. PMID:1187347

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

    PubMed

    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.

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

    PubMed

    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.

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

    SciTech Connect

    Lian, L.Y.; Yang, J.C.; Derrick, J.P.; Sutcliffe, M.J.; Roberts, G.C.K. ); Murphy, J.P.; Goward, C.R.; Atkinson, T. )

    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.

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

    PubMed

    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.

  8. Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy.

    PubMed

    Nagaraj, Madhu; Franks, Trent W; Saeidpour, Siavash; Schubeis, Tobias; Oschkinat, Hartmut; Ritter, Christiane; van Rossum, Barth-Jan

    2016-07-15

    Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT. PMID:27147408

  9. Multinuclear NMR studies of the flavodoxin from Anabaena 7120:. beta. -sheet structure and the flavin mononucleotide binding site

    SciTech Connect

    Stockman, B.J.

    1989-01-01

    A concerted approach to primary {sup 1}H, {sup 13}C, and {sup 15}N nuclear magnetic resonance assignments in proteins was developed. The method requires enrichment of the protein with {sup 13}C and {sup 15}N. The technique relies on the comparison of data sets from NMR experiments that correlate various nuclei: {sup 13}C({sup 13}C) double quantum correlations, {sup 1}H({sup 13}C) and {sup 1}H({sup 15}N) single bond correlations, and {sup 1}H({sup 13}C) and {sup 1}H({sup 15}N) multiple bond correlations. Comparison of data sets increases the number of resonances that can be assigned and improves assignment confidence. By combined use of these and conventional NMR techniques, sequential assignments were made for the {beta}-sheet and flavin mononucleotide (FMN) binding site residues in flavodoxin from Anabaena 7120. The {beta}-sheet structure was found to be similar to that seen in the crystal structure of Anacystis nidulans flavodoxin. In the FMN binding site, a total of 69 NOEs were identified: eight between protons of FMN, 36 between protons of binding site residues, and 25 between protons of FMN and protein. These constraints were used to determine the localized solution structure of the flavin binding site. The electronic environment and conformation of the protein-bound isoalloxazine ring were investigated by determining chemical shifts and coupling constants for the ring atoms. The carbonyl edge of the flavin ring was found to be slightly polarized by hydrogen bonding to the protein. The xylene ring was found to be nonplanar. The C{sup 6}-N{sup 5} region of the flavin appears to be solvent accessible.

  10. Molecular Details of Acetate Binding to a New Diamine Receptor by NMR and FT-IR Analyses.

    PubMed

    Banerji, Biswadip; Chatterjee, Moumita; Pal, Uttam; Maiti, Nakul Chandra

    2016-04-21

    Acetate anion plays an important role in several biochemical functions such as enzyme reaction, antibody response, and action of receptor molecules. This investigation reports the synthesis and molecular details of a unique receptor, 2-amino-N-(2-amino-benzyl)-benzamide (R) that senses selectively acetate via simultaneous involvement of one aromatic amine group and an amide proton of the receptor molecule. Solution-state NMR, steady-state fluorescence, and FT-IR examinations established that the acetate anion binds to the receptor with 1:1 ratio with high specificity. The binding was stabilized by two H-bond formations between the oxygen atoms of acetate anion and two H atoms, one from amide group and the other from the amine group of the receptor. The binding interaction caused significant changes in the chemical shift of the receptor protons, and the evaluated affinity constant, from the NMR measurements, was found to be 1.87 × 10(4) M(-1). Density functional theory (DFT) analysis further showed a significant rotation of one of the two aromatic rings leading to formation of a 10-member ring involving the acetate anion, amide proton, and the one amine group attached to aromatic ring. The H-bond patterns observed in the crystal structure were significantly changed due to complex formation. However, the changes in the geometrical arrangement in the complex caused a small but significant increase of the fluorescence emission. Acetate geometry and unique positioning of the amide and amine groups of the receptor render the recognition feasible, and DFT analysis estimated ∼30 kJ M(-1) stabilization due to 1:1 complexation. Such positioning and geometrical arrangement may make the receptor very specific to bind acetate anion, and as such R became a very relevant molecule in detection and function of the acetate anion present in complex biochemical systems.

  11. Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies.

    PubMed

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

    2016-09-15

    In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA.

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

    PubMed

    Davis, Craig Warren; Di Toro, Dominic M

    2015-07-01

    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. PMID:26035017

  13. Binding constant determination of uranyl-citrate complex by ACE using a multi-injection method.

    PubMed

    Zhang, Yiding; Li, Linnan; Huang, Hexiang; Xu, Linnan; Li, Ze; Bai, Yu; Liu, Huwei

    2015-04-01

    The binding constant determination of uranyl with small-molecule ligands such as citric acid could provide fundamental knowledge for a better understanding of the study of uranyl complexation, which is of considerable importance for multiple purposes. In this work, the binding constant of uranyl-citrate complex was determined by ACE. Besides the common single-injection method, a multi-injection method to measure the electrophoretic mobility was also applied. The BGEs used contained HClO4 and NaClO4 , with a pH of 1.98 ± 0.02 and ionic strength of 0.050 mol/L, then citric acid was added to reach different concentrations. The electrophoretic mobilities of the uranyl-citrate complex measured by both of the two methods were consistent, and then the binding constant was calculated by nonlinear fitting assuming that the reaction had a 1:1 stoichiometry and the complex was [(UO2 )(Cit)](-) . The binding constant obtained by the multi-injection method was log K = 9.68 ± 0.07, and that obtained by the single-injection method was log K = 9.73 ± 0.02. The results provided additional knowledge of the uranyl-citrate system, and they demonstrated that compared with other methods, ACE using the multi-injection method could be an efficient, fast, and simple way to determine electrophoretic mobilities and to calculate binding constants. PMID:25598434

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

    PubMed

    Davis, Craig Warren; Di Toro, Dominic M

    2015-07-01

    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.

  15. A new tripodal receptor for molecular recognition of monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy.

    PubMed

    Vacca, Alberto; Nativi, Cristina; Cacciarini, Martina; Pergoli, Roberto; Roelens, Stefano

    2004-12-22

    A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.

  16. 31P{1H}NMR and carbonyl force constants of unsymmetrical bidentate phosphine complexes of group (VI) metal carbonyls

    NASA Astrophysics Data System (ADS)

    Jesu Raj, Joe Gerald; Pathak, Devendra Deo; Kapoor, Pramesh N.

    2015-05-01

    In our present work we report synthesis of an unsymmetrical diphos ligand, 1-diphenylphosphino-2-di-m-tolylphosphinoethane and its coordinate complexes with group (VI) metal carbonyls such as Cr(CO)6 Mo(CO)6 and W(CO)6. The synthesized ligand and its complexes have been completely characterized by elemental analyses, FTIR, 1HNMR, 31P{1H}NMR and FAB mass spectrometry methods. Special emphasis has been given to calculations of carbonyl force constants. Based on the spectroscopic evidences it has been confirmed that these metal carbonyl complexes with the ditertiary phosphine ligand showed cis geometry in their molecular structure.

  17. Specific Lipid Binding of Membrane Proteins in Detergent Micelles Characterized by NMR and Molecular Dynamics.

    PubMed

    Zhao, Linlin; Wang, Shuqing; Run, Changqing; OuYang, Bo; Chou, James J

    2016-09-27

    Many membrane proteins bind specifically to lipids as an integral component of their structures. The ability of detergents to support lipid binding is thus an important consideration when solubilizing membrane proteins for structural studies. In particular, the zwitterionic phosphocholine (PC)-based detergents, which have been widely used in solution NMR studies of channels and transporters, are controversial because of their strong solubilization power and thus perceived as more denaturing than nonionic detergents such as the maltosides. Here, we investigate the ability of the mitochondrial ADP/ATP carrier (AAC) to specifically bind cardiolipin, a mitochondrial lipid important for the carrier function, in dodecylphosphocholine (DPC) micelles. We found that in DPC, the AAC specifically binds cardiolipin in a manner consistent with the bound cardiolipins found in the crystal structures of the AAC determined in n-decyl β-d-maltoside. Our results suggest that PC detergent is compatible with specific lipid binding and that PC detergent mixed with the relevant lipid represents a viable solubilization system for NMR studies of membrane proteins. PMID:27625145

  18. The Use of Hammett Constants to Understand the Non-Covalent Binding of Aromatics

    PubMed Central

    Lewis, Michael; Bagwill, Christina; Hardebeck, Laura K. E.; Wireduaah, Selina

    2012-01-01

    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. PMID:24688634

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

    SciTech Connect

    Demissie, Taye B.

    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.

  20. Anomalously slow cyanide binding to Glycera dibranchiata monomer methemoglobin component II: Implication for the equilibrium constant

    SciTech Connect

    Mintorovitch, J.; Satterlee, J.D. )

    1988-10-18

    In comparison to sperm whale metmyoglobin, metleghemoglobin {alpha}, methemoglobins, and heme peroxidases, the purified Glycera dibranchiata monomer methemoglobin component II exhibits anomalously slow cyanide ligation kinetics. For the component II monomer methemoglobin this reaction has been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0, employing 100-250-fold mole excesses of potassium cyanide at each pH. The analysis shows that the concentration-independent bimolecular rate constant is small in comparison to those of the other heme proteins. Furthermore, the results show that the dissociation rate is extremely slow. Separation of the bimolecular rate constant into contributions from k{sub CN{sup {minus}}} (the rate constant for CN{sup {minus}} binding) and from k{sub HCN} (the rate constant for HCN binding) shows that the former is approximately 90 times greater. These results indicate that cyanide ligation reactions are not instantaneous for this protein, which is important for those attempting to study the ligand-binding equilibria. From the results presented here the authors estimate that the actual equilibrium dissociation constant (K{sub D}) for cyanide binding to this G. dibranchiata monomer methemoglobin has a numerical upper limit that is at least 2 orders of magnitude smaller than the value reported before the kinetic results were known.

  1. Equilibrium binding constants for Tl+ with gramicidins A, B and C in a lysophosphatidylcholine environment determined by 205Tl nuclear magnetic resonance spectroscopy.

    PubMed Central

    Hinton, J F; Koeppe, R E; Shungu, D; Whaley, W L; Paczkowski, J A; Millett, F S

    1986-01-01

    Nuclear Magnetic Resonance (NMR) 205Tl spectroscopy has been used to monitor the binding of Tl+ to gramicidins A, B, and C packaged in aqueous dispersions of lysophosphatidylcholine. For 5 mM gramicidin dimer in the presence of 100 mM lysophosphatidylcholine, only approximately 50% or less of the gramicidin appears to be accessible to Tl+. Analysis of the 205Tl chemical shift as a function of Tl+ concentration over the 0.65-50 mM range indicates that only one Tl+ ion can be bound by gramicidin A, B, or C under these experimental conditions. In this system, the Tl+ equilibrium binding constant is 582 +/- 20 M-1 for gramicidin 1949 +/- 100 M-1 for gramicidin B, and 390 +/- 20 M-1 for gramicidin C. Gramicidin B not only binds Tl+ more strongly but it is also in a different conformational state than that of A and C, as shown by Circular Dichroism spectroscopy. The 205Tl NMR technique can now be extended to determinations of binding constants of other cations to gramicidin by competition studies using a 205Tl probe. PMID:2420383

  2. Solid-State NMR Characterization of Mixed Phosphonic Acid Ligand Binding and Organization on Silica Nanoparticles.

    PubMed

    Davidowski, Stephen K; Holland, Gregory P

    2016-04-01

    As ligand functionalization of nanomaterials becomes more complex, methods to characterize the organization of multiple ligands on surfaces is required. In an effort to further the understanding of ligand-surface interactions, a combination of multinuclear ((1)H, (29)Si, (31)P) and multidimensional solid-state nuclear magnetic resonance (NMR) techniques was utilized to characterize the phosphonic acid functionalization of fumed silica nanoparticles using methylphosphonic acid (MPA) and phenylphosphonic acid (PPA). (1)H → (29)Si cross-polarization (CP)-magic angle spinning (MAS) solid-state NMR was used to selectively detect silicon atoms near hydrogen atoms (primarily surface species); these results indicate that geminal silanols are preferentially depleted during the functionalization with phosphonic acids. (1)H → (31)P CP-MAS solid-state NMR measurements on the functionalized silica nanoparticles show three distinct resonances shifted upfield (lower ppm) and broadened compared to the resonances of the crystalline ligands. Quantitative (31)P MAS solid-state NMR measurements indicate that ligands favor a monodentate binding mode. When fumed silica nanoparticles were functionalized with an equal molar ratio of MPA and PPA, the MPA bound the nanoparticle surface preferentially. Cross-peaks apparent in the 2D (1)H exchange spectroscopy (EXSY) NMR measurements of the multiligand sample at short mixing times indicate that the MPA and PPA are spatially close (≤5 Å) on the surface of the nanostructure. Furthermore, (1)H-(1)H double quantum-single quantum (DQ-SQ) back-to-back (BABA) 2D NMR spectra further confirmed that MPA and PPA are strongly dipolar coupled with observation of DQ intermolecular contacts between the ligands. DQ experimental buildup curves and simulations indicate that the average distance between MPA and PPA is no further than 4.2 ± 0.2 Å. PMID:26914738

  3. Proteolytic footprinting titrations for estimating ligand-binding constants and detecting pathways of conformational switching of calmodulin.

    PubMed

    Shea, M A; Sorensen, B R; Pedigo, S; Verhoeven, A S

    2000-01-01

    To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized "silent" interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibirum titrations monitored by NMR. By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one

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

    SciTech Connect

    Fagan, P A

    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{center_dot}C base pairs are functional analogs of A{center_dot}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.

  5. CONSTANTS FOR MERCURY BINDING BY DISSOLVED ORGANIC MATTER ISOLATES FROM THE FLORIDA EVERGLADES. (R827653)

    EPA Science Inventory

    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 appro...

  6. Kinetic rate constant prediction supports the conformational selection mechanism of protein binding.

    PubMed

    Moal, Iain H; Bates, Paul A

    2012-01-01

    The prediction of protein-protein kinetic rate constants provides a fundamental test of our understanding of molecular recognition, and will play an important role in the modeling of complex biological systems. In this paper, a feature selection and regression algorithm is applied to mine a large set of molecular descriptors and construct simple models for association and dissociation rate constants using empirical data. Using separate test data for validation, the predicted rate constants can be combined to calculate binding affinity with accuracy matching that of state of the art empirical free energy functions. The models show that the rate of association is linearly related to the proportion of unbound proteins in the bound conformational ensemble relative to the unbound conformational ensemble, indicating that the binding partners must adopt a geometry near to that of the bound prior to binding. Mirroring the conformational selection and population shift mechanism of protein binding, the models provide a strong separate line of evidence for the preponderance of this mechanism in protein-protein binding, complementing structural and theoretical studies.

  7. Unraveling the Conformational Landscape of Ligand Binding to Glucose/Galactose-Binding Protein by Paramagnetic NMR and MD Simulations.

    PubMed

    Unione, Luca; Ortega, Gabriel; Mallagaray, Alvaro; Corzana, Francisco; Pérez-Castells, Javier; Canales, Angeles; Jiménez-Barbero, Jesús; Millet, Oscar

    2016-08-19

    Protein dynamics related to function can nowadays be structurally well characterized (i.e., instances obtained by high resolution structures), but they are still ill-defined energetically, and the energy landscapes are only accessible computationally. This is the case for glucose-galactose binding protein (GGBP), where the crystal structures of the apo and holo states provide structural information for the domain rearrangement upon ligand binding, while the time scale and the energetic determinants for such concerted dynamics have been so far elusive. Here, we use GGBP as a paradigm to define a functional conformational landscape, both structurally and energetically, by using an innovative combination of paramagnetic NMR experiments and MD simulations. Anisotropic NMR parameters induced by self-alignment of paramagnetic metal ions was used to characterize the ensemble of conformations adopted by the protein in solution while the rate of interconversion between conformations was elucidated by long molecular dynamics simulation on two states of GGBP, the closed-liganded (holo_cl) and open-unloaded (apo_op) states. Our results demonstrate that, in its apo state, the protein coexists between open-like (68%) and closed-like (32%) conformations, with an exchange rate around 25 ns. Despite such conformational heterogeneity, the presence of the ligand is the ultimate driving force to unbalance the equilibrium toward the holo_cl form, in a mechanism largely governed by a conformational selection mechanism. PMID:27219646

  8. Non-empirical calculations of NMR indirect carbon-carbon coupling constants. Part 6: propellanes.

    PubMed

    Krivdin, Leonid B

    2004-01-01

    A full set of carbon-carbon coupling constants have been calculated at the SOPPA level in the series of six most representative propellanes. Special attention was focused on spin-spin couplings involving both bridgehead carbons, and these data were rationalized in terms of the multipath coupling mechanism and hybridization effects. Many unknown couplings in the propellane frameworks were predicted with high reliability.

  9. NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10.

    PubMed

    Künze, Georg; Gehrcke, Jan-Philip; Pisabarro, M Teresa; Huster, Daniel

    2014-11-01

    The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

  10. BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

    PubMed Central

    Aguirre, Clémentine; ten Brink, Tim; Walker, Olivier; Guillière, Florence; Davesne, Dany; Krimm, Isabelle

    2013-01-01

    Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach. PMID:23717610

  11. High-Throughput Phase-Distribution Method to Determine Drug-Cyclodextrin Binding Constants

    PubMed Central

    CHEN, ZHI; LU, DUJUAN; WEBER, STEPHEN G.

    2009-01-01

    A high-throughput method has been developed to measure drug-cyclodextrin binding constants. It measures the distribution ratio of a drug between a polymer film [polyvinyl chloride (PVC) with 67% (w/w) dioctyl sebacate (DOS)] and a cyclodextrin-containing buffer in a 96-well format. Measurements of distribution ratios at several cyclodextrin concentrations lead to binding constants. Binding constants for econazole with six CDs have been determined in one 96-well microplate with four replications of each condition in 10 h. The K1:1/103 M−1 values are 3.98±0.13, 3.90±0.22, 29.3±2.2, 0.66±0.04 1.78±0.30, 4.08±0.50, with (2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, hepta-kis(2,3,6-tri-O-methyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin, respectively. It is likely that 1:2 complexes are also formed in some cases. This method has also been applied to study the binding behavior as a function of the drug concentration and pH. Binding weakens at higher drug concentration which may be due to the self-association of the drug. An acidic environment decreases the binding constant of CD with the basic econazole. The formation of the 1:2 complexes is completely suppressed in acid as well. This protocol is faster than the phase-solubility method. Moreover, the material requirement is up to four orders of magnitude lower. PMID:18428984

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

    PubMed

    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.

  13. NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity

    SciTech Connect

    Roberts, Arthur G.; Diaz, Maria D.; Lampe, Jed N.; Shireman, Laura; Grinstead, Jeffrey S.; Dabrowski, Michael J.; Pearson, Josh T.; Bowman, Michael K.; Atkins, William M.; Campbell, Ann P.

    2006-02-14

    Cytochrome P450’s (P450’s) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450’s demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450eryF. UV/vis absorbance spectra of P450eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS ) 4 and 200 íM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hillequation- derived KS values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands “contact” the same phenylalanines within the active site of P450eryF. This finding is in agreement with X-ray crystal structures of bound P450eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the lowaffinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.

  14. Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy

    PubMed Central

    Drögemüller, Johanna; Strauß, Martin; Schweimer, Kristian; Jurk, Marcel; Rösch, Paul; Knauer, Stefan H.

    2015-01-01

    In bacteria, RNA polymerase (RNAP), the central enzyme of transcription, is regulated by N-utilization substance (Nus) transcription factors. Several of these factors interact directly, and only transiently, with RNAP to modulate its function. As details of these interactions are largely unknown, we probed the RNAP binding surfaces of Escherichia coli (E. coli) Nus factors by nuclear magnetic resonance (NMR) spectroscopy. Perdeuterated factors with [1H,13C]-labeled methyl groups of Val, Leu, and Ile residues were titrated with protonated RNAP. After verification of this approach with the N-terminal domain (NTD) of NusG and RNAP we determined the RNAP binding site of NusE. It overlaps with the NusE interaction surface for the NusG C-terminal domain, indicating that RNAP and NusG compete for NusE and suggesting possible roles for the NusE:RNAP interaction, e.g. in antitermination and direct transcription:translation coupling. We solved the solution structure of NusA-NTD by NMR spectroscopy, identified its RNAP binding site with the same approach we used for NusG-NTD, and here present a detailed model of the NusA-NTD:RNAP:RNA complex. PMID:26560741

  15. NMR Characterizations of the Ice Binding Surface of an Antifreeze Protein

    PubMed Central

    Li, Congmin; Jia, Zongchao; Xia, Bin; Jin, Changwen

    2010-01-01

    Antifreeze protein (AFP) has a unique function of reducing solution freezing temperature to protect organisms from ice damage. However, its functional mechanism is not well understood. An intriguing question concerning AFP function is how the high selectivity for ice ligand is achieved in the presence of free water of much higher concentration which likely imposes a large kinetic barrier for protein-ice recognition. In this study, we explore this question by investigating the property of the ice binding surface of an antifreeze protein using NMR spectroscopy. An investigation of the temperature gradient of amide proton chemical shift and its correlation with chemical shift deviation from random coil was performed for CfAFP-501, a hyperactive insect AFP. A good correlation between the two parameters was observed for one of the two Thr rows on the ice binding surface. A significant temperature-dependent protein-solvent interaction is found to be the most probable origin for this correlation, which is consistent with a scenario of hydrophobic hydration on the ice binding surface. In accordance with this finding, rotational correlation time analyses combined with relaxation dispersion measurements reveals a weak dimer formation through ice binding surface at room temperature and a population shift of dimer to monomer at low temperature, suggesting hydrophobic effect involved in dimer formation and hence hydrophobic hydration on the ice binding surface of the protein. Our finding of hydrophobic hydration on the ice binding surface provides a test for existing simulation studies. The occurrence of hydrophobic hydration on the ice binding surface is likely unnecessary for enhancing protein-ice binding affinity which is achieved by a tight H-bonding network. Subsequently, we speculate that the hydrophobic hydration occurring on the ice binding surface plays a role in facilitating protein-ice recognition by lowering the kinetic barrier as suggested by some simulation

  16. Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

    SciTech Connect

    Barden, J.A.

    1987-09-22

    The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I ..beta..-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.

  17. The role of propionates in substrate binding to heme oxygenase from Neisseria meningitidis; A NMR study†

    PubMed Central

    Peng, Dungeng; Ma, Li-Hua; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

    2012-01-01

    Heme oxygenase, HO, cleaves hemin into biliverdin, iron and CO. For mammalian HOs, both native hemin propionates are required for substrate binding and activity. The HO from the pathogenic bacterium Neisseria meningitidis, NmHO, possesses a crystallographically undetected C-terminal fragment that by solution 1H NMR is found to fold and interact with the active site. One of the substrate propionates has been proposed to form a salt bridge to the C-terminus rather than to the conventional buried cationic side chain in other HOs. Moreover, the C-terminal dipeptide Arg208His209 cleaves spontaneously over ~24 hours at a rate dependent on substituent size. 2D 1H NMR of NmHO azide complexes with hemins with selectively deleted or rearranged propionates all bind to NmHO with a structurally conserved active site as reflected in optical spectra and NMR NOESY cross peak and hyperfine shift patterns. In contrast to mammalian HOs, NmHO requires only a single propionate interacting with the buried terminus of Lys16 to exhibit full activity and tolerates the existence of a propionate at the exposed 8-position. The structure of the C-terminus is qualitatively retained upon deletion of the 7-propionate but a dramatic change in the 7-propionate carboxylate 13C chemical shift upon C-terminal cleavage confirms its role in the interaction with the C-terminus. The stronger hydrophobic contacts between pyrroles A and B with NmHO contribute more substantially to the substrate binding free energy than in mammalian HOs, “liberating” one propionate to stabilize the C-terminus. The functional implications of the C-terminus in product release are discussed. PMID:22913621

  18. Non-empirical calculations of NMR indirect carbon-carbon coupling constants. Part 7--spiroalkanes.

    PubMed

    Krivdin, Leonid B

    2004-06-01

    Carbon-carbon spin-spin coupling constants were calculated at the SOPPA level for a series of seven classical spiroalkanes, spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, spiro[3.3]heptane, spiro[4.4]nonane and spiro[5.5]undecane, with special focus upon couplings involving and/or across spiro carbons. Many interesting structural trends were investigated originating in specific geometries and unusual bonding environments at the spiro carbon.

  19. Rationalizing 5000-fold differences in receptor-binding rate constants of four cytokines.

    PubMed

    Pang, Xiaodong; Qin, Sanbo; Zhou, Huan-Xiang

    2011-09-01

    The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as k(a)=k(a0)exp(-ΔG(el)(∗)/k(B)T) where k(a0) is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (k(a0)) of EPO, IL4, hGH, and PRL were similar (5.2 × 10(5) M(-1)s(-1), 2.4 × 10(5) M(-1)s(-1), 1.7 × 10(5) M(-1)s(-1), and 1.7 × 10(5) M(-1)s(-1), respectively). However, the average electrostatic free energies (ΔG(e1)(∗)) were very different (-4.2 kcal/mol, -2.4 kcal/mol, -0.1 kcal/mol, and -0.5 kcal/mol, respectively, at ionic strength=160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 10(8) M(-1)s(-1), 1.3 × 10(7) M(-1)s(-1), 2.0 × 10(5) M(-1)s(-1), and 7.6 × 10(4) M(-1)s(-1), respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps

  20. Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.

    PubMed

    Wang, Jing; Rabenstein, Dallas L

    2007-09-01

    Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.

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

    NASA Astrophysics Data System (ADS)

    Faber, Rasmus; Sauer, Stephan P. A.

    2015-12-01

    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.

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

    SciTech Connect

    Faber, Rasmus; Sauer, Stephan P. A.

    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.

  3. Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

    SciTech Connect

    Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie; Tiralongo, Joe; Gerardy-Schahn, Rita; Itzstein, Mark von . E-mail: m.vonitzstein@griffith.edu.au

    2007-08-10

    We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand ({alpha}/{beta}-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.

  4. Purification and stable isotope labeling of the calcium- and integrin-binding protein 1 for structural and functional NMR studies.

    PubMed

    Huang, Hao; Vogel, Hans J

    2013-01-01

    The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit. In this protocol, we describe the purification and isotope labeling strategies for productive NMR studies of CIB1. The same isotope labeling strategies can be implemented to study numerous related regulatory calcium-binding proteins.

  5. Determination of binding constants by affinity capillary electrophoresis, electrospray ionization mass spectrometry and phase-distribution methods

    PubMed Central

    Chen, Zhi; Weber, Stephen G.

    2008-01-01

    Many methods for determining intermolecular interactions have been described in the literature in the past several decades. Chief among them are methods based on spectroscopic changes, particularly those based on absorption or nuclear magnetic resonance (NMR) [especially proton NMR (1H NMR)]. Recently, there have been put forward several new methods that are particularly adaptable, use very small quantities of material, and do not place severe requirements on the spectroscopic properties of the binding partners. This review covers new developments in affinity capillary electrophoresis, electrospray ionization mass spectrometry (ESI-MS) and phasetransfer methods. PMID:19802330

  6. Proton zero-quantum 2D NMR of 2-propenenitrile aligned by an electric field. Determination of the 2H and 14N quadrupole coupling constants

    NASA Astrophysics Data System (ADS)

    Ruessink, B. H.; De Kanter, F. J. J.; MaClean, C.

    Zero-quantum NMR, selectively detected by 2D NMR, is applied to observe small 1H- 1H dipolar couplings in a polar liquid partially oriented by a strong electric field. The normal (single-quantum) 1H spectrum is severely broadened, which prevents the observation of small couplings. The results from the zero-quantum proton spectrum are used to calculate the 2H and 14N quadrupole coupling constants of 2-deutero-2-propenenitrile from the 2H and 14N NMR spectra.

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

    PubMed

    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. PMID:22887687

  8. Binding mode characterization of novel RNA polymerase inhibitors using a combined biochemical and NMR approach.

    PubMed

    Fruth, Martina; Plaza, Alberto; Hinsberger, Stefan; Sahner, Jan Henning; Haupenthal, Jörg; Bischoff, Markus; Jansen, Rolf; Müller, Rolf; Hartmann, Rolf W

    2014-11-21

    Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant Staphylococcus aureus (MRSA) strains (MIC 2-16 μg mL(-1)) and rifampicin-resistant Escherichia coli TolC strains (MIC 12.5-50 μg mL(-1)). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (Myx) suggest that the ureidothiophene-2-carboxylic acids compete with Myx for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the Myx western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance.

  9. Does the ligand-biopolymer equilibrium binding constant depend on the number of bound ligands?

    PubMed

    Beshnova, Daria A; Lantushenko, Anastasia O; Evstigneev, Maxim P

    2010-11-01

    Conventional methods, such as Scatchard or McGhee-von Hippel analyses, used to treat ligand-biopolymer interactions, indirectly make the assumption that the microscopic binding constant is independent of the number of ligands, i, already bound to the biopolymer. Recent results on the aggregation of aromatic molecules (Beshnova et al., J Chem Phys 2009, 130, 165105) indicated that the equilibrium constant of self-association depends intrinsically on the number of molecules in an aggregate due to loss of translational and rotational degrees of freedom on formation of the complex. The influence of these factors on the equilibrium binding constant for ligand-biopolymer complexation was analyzed in this work. It was shown that under the conditions of binding of "small" molecules, these factors can effectively be ignored and, hence, do not provide any hidden systematic error in such widely-used approaches, such as the Scatchard or McGhee-von Hippel methods for analyzing ligand-biopolymer complexation. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 932-935, 2010.

  10. A rigorous multiple independent binding site model for determining cell-based equilibrium dissociation constants.

    PubMed

    Drake, Andrew W; Klakamp, Scott L

    2007-01-10

    A new 4-parameter nonlinear equation based on the standard multiple independent binding site model (MIBS) is presented for fitting cell-based ligand titration data in order to calculate the ligand/cell receptor equilibrium dissociation constant and the number of receptors/cell. The most commonly used linear (Scatchard Plot) or nonlinear 2-parameter model (a single binding site model found in commercial programs like Prism(R)) used for analysis of ligand/receptor binding data assumes only the K(D) influences the shape of the titration curve. We demonstrate using simulated data sets that, depending upon the cell surface receptor expression level, the number of cells titrated, and the magnitude of the K(D) being measured, this assumption of always being under K(D)-controlled conditions can be erroneous and can lead to unreliable estimates for the binding parameters. We also compare and contrast the fitting of simulated data sets to the commonly used cell-based binding equation versus our more rigorous 4-parameter nonlinear MIBS model. It is shown through these simulations that the new 4-parameter MIBS model, when used for cell-based titrations under optimal conditions, yields highly accurate estimates of all binding parameters and hence should be the preferred model to fit cell-based experimental nonlinear titration data. PMID:17141800

  11. A two-constant equation for multiple albumin-binding isotherms.

    PubMed

    Larsen, C G; Larsen, F G; Brodersen, R

    1986-07-01

    It has been found that binding of low molecular weight ligands to human serum albumin is generally nonsaturating. Equations commonly used for describing the binding equilibria, i.e., the Scatchard and Klotz (Adair) equations, are saturation functions. We have accordingly tried to establish an equation which would fit the observed data, i.e., not reach a saturation plateau. An empirical equation, in which the bound ligand is expressed as a function of the free ligand [R(C) = b1 in (b2C + 1)], is shown to give reasonably good fits to observed binding equilibrium data for the binding of several organic ligands to human serum albumin, when the two parameters, b1 and b2, are given suitable values. The curve of bound versus free ligand, as plotted from this equation, has the same slope and curvature as that obtained from the Klotz stepwise binding equation at C = 0, if b1 = K1/[2(K1 - 2K2)] and b2 = 2(K1 - 2K2), where K1 and K2 are the first and second stoichiometric binding constants.

  12. Probing the flexibility of internal rotation in silylated phenols with the NMR scalar spin-spin coupling constants.

    PubMed

    Sychrovský, Vladimír; Benda, Ladislav; Prokop, Alexandr; Blechta, Vratislav; Schraml, Jan; Spirko, Vladimír

    2008-06-12

    The rotation of a trimethylsiloxy (TMSO) group in three silylated phenols (with three different ortho substituents -H, -CH3, and -C(CH3)3) was studied with the NMR (n)J(Si,C), n = 2, 3, 4, 5, scalar spin-spin coupling between the (29)Si nucleus of the TMSO group and the (13)C nuclei of the phenyl ring. The internal rotation potential calculated with the B3LYP and MP2 calculation methods including the effect of a solvent environment (gas phase, chloroform, and water) was used for the calculation of the dynamical averages of the scalar coupling constants in the framework of the rigid-bender formalism. Solvent effects, the quality of the rotational potential, and the applicability of the classical molecular dynamic to the problem is discussed. Quantum effects have a sizable impact on scalar couplings, particularly for the internal rotational states well localized within the wells of the potential surfaces for the TMSO group. The overall difference between the experimental and theoretical scalar couplings calculated for the global energy-minima structures (static model) decreases substantially for both model potentials (B3LYP, MP2) when the molecular motion of the TMSO group is taken into account. The calculated data indicate that the inclusion of molecular motion is necessary for the accurate calculation of the scalar coupling constants and their reliable structural interpretation for any system which possesses a large-amplitude motion. PMID:18491850

  13. Experimental, SOPPA(CCSD), and DFT analysis of substitutent effects on NMR 1JCF coupling constants in fluorobenzene derivatives.

    PubMed

    Vilcachagua, Janaina Dantas; Ducati, Lucas C; Rittner, Roberto; Contreras, Rubén H; Tormena, Cláudio F

    2011-02-24

    Interesting insight into the electronic molecular structure changes associated with substituent effects on the Fermi contact (FC) and paramagnetic spin-orbit (PSO) terms of (1)J(CF) NMR coupling constants (SSCCs) in o-X-, m-X-, and p-X-fluorobenzenes (X = NH(2); NO(2)) is presented. The formulation of this approach is based on the influence of different conjugative and hyperconjugative interactions on a second-order property, which can be qualitatively predicted if it is known how they affect the main virtual excitations entering into that second-order property. A set of consistent approximations are introduced in order to analyze the behavior of occupied and virtual orbitals, which define some experimental trends for (1)J(CF) spin-spin coupling constants. In addition, DFT hybrid functionals were used, and a similar degree of confidence to compute the (1)J(CF) with those observed for the SOPPA(CCSD) method was obtained. The (1)J(CF) SSCCs for ezetimibe, a commercially fluorinated drug used to reduce cholesterol levels, were measured and DFT-calculated, and the qualitative approach quoted above was applied. As a byproduct, a possible method to determine experimentally a significant PSO contribution to (1)J(CF) SSCCs is discussed.

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

    USGS Publications Warehouse

    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.

  15. 13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin.

    PubMed

    Bjornson, M E; Corson, D C; Sykes, B D

    1985-10-01

    13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.

  16. Calculation of cooperativity and equilibrium constants of ligands binding to G-quadruplex DNA in solution.

    PubMed

    Kudrev, A G

    2013-11-15

    Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption onto a lattice of multiple binding sites. An experimental example has been used to verify the assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. To evaluate a ligand binding affinity and cooperativity of ligand-oligomer complex formation the statistical approach has been proposed. This new computational approach used to re-examine previously studded ligand binding towards DNA quadruplexes targets with multiple binding sites. The intrinsic equilibrium constants K1-3 of the mesotetrakis-(N-methyl-4-pyridyl)-porphyrin (TMPyP4) binding with the [d(T4G4)]4 and with the [AG3(T2AG3)3] quadruplexes and the correction for the mutual influence between bound ligands (cooperativity parameters ω) was determined from the Job plots based upon the nonlinear least-squares fitting procedure. The re-examination of experimental curves reveals that the equilibrium is affected by the positive cooperative (ω>1) binding of the TMPyP4 ligand with tetramolecular [d(T4G4)]4. However for an intramolecular antiparallel-parallel hybrid structure [AG3(T2AG3)3] the weak anti-cooperativity of TMPyP4 accommodation (ω<1) onto two from three nonidentical sites was detected. PMID:24148442

  17. Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

    PubMed Central

    Dani, J A; Levitt, D G

    1981-01-01

    In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+. PMID:6168310

  18. NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c-di-GMP binding protein

    SciTech Connect

    Ramelot, Theresa A; Yee, Adelinda; Cort, John R; Semesi, Anthony; Arrowsmith, Cheryl H; Kennedy, Michael A

    2007-02-01

    PA4608 is a 125 residue protein with a proposed identification as a PilZ domain and c-di-GMP adaptor protein that plays a role in bacterial second-messenger regulated processes. The NMR structure of PA4608 has been determined and c-di-GMP binding has been confirmed by NMR titration studies. The monomeric structure of PA4608 contains a six-stranded anti-parallel β barrel flanked by three helices. Conserved surface residues among PA4608 homologs suggest the the c-di-GMP binding site is at one end of the barrel and includes residues in the helices as well as in the unstructured N-terminus. Chemical shift changes upon binding to c-di-GMP confirm that PA4608 binds to c-di-GMP. This evidence supports the hypothesis that proteins containing PilZ domains are the long-sought c-di-GMP adaptor proteins.

  19. Quantum electrodynamics effects on NMR magnetic shielding constants of He-like and Be-like atomic systems

    NASA Astrophysics Data System (ADS)

    Gimenez, Carlos A.; Kozioł, Karol; Aucar, Gustavo A.

    2016-03-01

    NMR shielding constants for He- and Be-like atomic systems of Ne, Ar, Kr, Xe, and Rn have been calculated at the random-phase-approximation level of approach, including an estimation of QED corrections within the polarization propagator formalism. We show that QED effects enhance electron correlation when Z becomes heavier, which happens with relativistic effects, and also that QED effects become smaller when going from more to less ionized systems. We studied two- and four-electron systems. Then such studies could easily be generalized to other many-electron systems. Results of calculations with our relatively simple model, which includes QED and electron correlation effects on the same theoretical grounds, have a summarized error in the range from 10% (for Ne) up to 24% (for Rn), so that our accuracy is a little lower than for calculations on H-like systems. Our findings should stimulate the development and/or the application of more rigorous formalisms to get more accurate QED corrections to response properties in many-electron systems.

  20. Thallium-205 NMR determination of the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A and C incorporated into model phospholipid membranes: An equilibrium study

    SciTech Connect

    Fernandez, J.Q.

    1987-01-01

    Thermodynamic parameters have been determined for the binding of monovalent and divalent cations to gramicidin A and C incorporated in lyso-{alpha}-phosphatidylcholine dispersions. The thermodynamic analyses used equilibrium constants derived from the analysis of Tl-205 NMR chemical shifts using a one-site binding model of the gramicidin channel. Initial experiments determined the thermodynamic parameters of the Tl{sup +} ion binding to gramicidin A. Next, the competitive binding-Tl-205 NMR technique was used to obtain the thermodynamic parameters for the binding of Li{sup +}, Na{sup +}, K{sup +}, Rb{sup +}, Cs{sup +}, NH{sub 4}{sup +} and Ag{sup +} cations to gramicidin A. A similar experiment for the binding of the Li{sup +}, Na{sup +}, K{sup +} and Rb{sup +} cations to gramicidin C was performed. Similar experiments determined the thermodynamic parameter for the Cd{sup ++}, Mg{sup ++}, Ca{sup ++}, Sr{sup ++} and Ba{sup ++} cations. Thermodynamic parameters have also been determined for the binding of the Tl{sup +} ion to gramicidin A incorporated into dimyristoylphosphatidylcholine vesicles using Tl-205 NMR spectroscopy.

  1. Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.

    PubMed Central

    Crocker, P R; Vinson, M; Kelm, S; Drickamer, K

    1999-01-01

    The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins. PMID:10393093

  2. Covalent binding of aniline to humic substances. 2. 15N NMR studies of nucleophilic addition reactions

    USGS Publications Warehouse

    Thorn, K.A.; Pettigrew, P.J.; Goldenberg, W.S.; Weber, E.J.

    1996-01-01

    Aromatic amines are known to undergo covalent binding with humic substances in the environment. Although previous studies have examined reaction conditions and proposed mechanisms, there has been no direct spectroscopic evidence for the covalent binding of the amines to the functional groups in humic substances. In order to further elucidate the reaction mechanisms, the Suwannee River and IHSS soil fulvic and humic acids were reacted with 15N-labeled aniline at pH 6 and analyzed using 15N NMR spectrometry. Aniline underwent nucleophilic addition reactions with the quinone and other carbonyl groups in the samples and became incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, imine, and heterocyclic nitrogen, the latter comprising 50% or more of the bound amine. The anilide and anilinohydroquinone nitrogens were determined to be susceptible to chemical exchange by ammonia. In the case of Suwannee River fulvic acid, reaction under anoxic conditions and pretreatment with sodium borohydride or hydroxylamine prior to reaction under oxic conditions resulted in a decrease in the proportion of anilinohydroquinone nitrogen incorporated. The relative decrease in the incorporation of anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen under anoxic conditions suggested that inter- or intramolecular redox reactions accompanied the nucleophilic addition reactions.

  3. NMR insights into dynamics regulated target binding of DLC8 dimer

    SciTech Connect

    Krishna Mohan, P.M.; Hosur, Ramakrishna V. . E-mail: hosur@tifr.res.in

    2007-04-20

    Conformational dynamics play a crucial role in biological function. Dynein light chain protein (DLC8) acts as a cargo adaptor, and exists as a dimer under physiological conditions and dissociates into monomer below pH 4. In the present NMR study, we identified some dynamic residues in the dimer using chemical shift perturbation approach by applying small pH change. As evidenced by gel filtration and CD studies, this small pH change does not alter the globular structural features of the protein. In fact, these changes result in small local stability perturbations as monitored using temperature dependence of amide proton chemical shifts, and influence the dynamics of the dimer substantially. Further, interaction studies of the protein with a peptide containing the recognition motif of cargo indicated that the efficacy of peptide binding decreases when the pH is reduced from 7 to 6. These observations taken together support the conception that dynamics can regulate cargo binding/trafficking by the DLC8 dimer.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Association constants of Pb2+ with binding sites of fungal biomass using metal-based titrations.

    PubMed

    Naja, G; Mustin, C; Volesky, B; Berthelin, J

    2006-01-01

    Biosorption is perceived as an alternative method for toxic heavy metal removal/recovery from aqueous effluents. This work focused on derivation of some key quantitative physico-chemical characteristics of a representative biosorbent material required for its further effective exploitation. The newly developed acid-base and metal-based titrator allowed the characterization of the chemisorption active sites of Rhizopus arrhizus biomass and the study of their metal affinity. This experimental approach, combined with an analytical method consisting of transforming the initial data enabled the calculation of the number and capacity of the reactive sites (Qads) and the metal affinity constants (Km) for lead sorption by R. arrhizus biomass. The pKm values for Rhizopus biomass varied between -3 and -6 for sites releasing no protons, -1 and 1 for sites releasing one proton, and > 8 for sites releasing two protons - combined with the Pb precipitation phenomenon. At low temperatures, the active binding site number was lower at lower lead concentrations whereas the precipitation was promoted at higher lead concentration values. Lead adsorption contributed modestly (11%) to its overall uptake and occurred at low lead concentrations onto strong and medium affinity binding sites. Micro-precipitation quickly commenced around active binding sites distinguished by their weak affinity whenever the solution lead concentrations reached 10(-6) or 10(-5) M and represented more than 85% of the total sorbed metal quantity. The work also demonstrated the usefulnes of the methodology reported here for characterizing complex biosorbent materials.

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

    PubMed

    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.

  7. NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

    PubMed Central

    Aachmann, Finn L.; Sørlie, Morten; Skjåk-Bræk, Gudmund; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

    2012-01-01

    Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu2+ (Kd = 55 nM; from isothermal titration calorimetry) and higher preference for Cu1+ (Kd ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu2+ of 275 mV using a thermodynamic cycle). Strong binding of Cu1+ was also reflected in a reduction in the pKa values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a 2H/1H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates. PMID:23112164

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

    PubMed Central

    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

  9. DFT calculations of 15N NMR shielding constants, chemical shifts and complexation shifts in complexes of rhodium(II) tetraformate with some nitrogenous organic ligands

    NASA Astrophysics Data System (ADS)

    Leniak, Arkadiusz; Jaźwiński, Jarosław

    2015-03-01

    Benchmark calculations of 15N NMR shielding constants for a set of model complexes of rhodium(II) tetraformate with nine organic ligands using the Density Functional Theory (DFT) methods have been carried out. The calculations were performed by means of several methods: the non-relativistic, relativistic scalar ZORA, and spin-orbit ZORA approaches at the CGA-PBE/QZ4P theory level, and the GIAO NMR method using the B3PW91 functional with the 6-311++G(2d,p) basis set for C, H, N, O atoms and the Stuttgart basis set for the Rh atom. The geometry of compounds was optimised either by the same basis set as for the NMR calculations or applying the B3LYP functional with the 6-31G(2d) basis set for C, H, N, O atoms and LANL2DZ for the Rh atom. Computed 15N NMR shielding constants σ were compatible with experimental 15N chemical shifts δ of complexes exhibiting similar structure and fulfil the linear equation δ = aσ + b. The a and b parameters for all data sets have been estimated by means of linear regression analysis. In contrast to the correlation method giving "scaled" chemical shifts, the conversion of shielding constants to chemical shifts with respect to the reference shielding of CH3NO2 provided very inaccurate "raw" δ values. The application of the former to the calculation of complexation shifts Δδ (Δδ = δcompl - δlig) reproduced experimental values qualitatively or semi-quantitatively. The non-relativistic B3PW91/[6-311++G(2d,p), Stuttgart] theory level reproduced the NMR parameters as good as the more expensive relativistic CGA-PBE//QZ4P ZORA approaches.

  10. A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

    PubMed

    Insaidoo, Francis K; Zajicek, Jaroslav; Baker, Brian M

    2009-10-20

    T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism.

  11. A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

    PubMed

    Insaidoo, Francis K; Zajicek, Jaroslav; Baker, Brian M

    2009-10-20

    T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism. PMID:19772349

  12. Defining the intramembrane binding mechanism of sarcolipin to calcium ATPase using solution NMR spectroscopy.

    PubMed

    Buffy, Jarrod J; Buck-Koehntop, Bethany A; Porcelli, Fernando; Traaseth, Nathaniel J; Thomas, David D; Veglia, Gianluigi

    2006-04-28

    Sarcolipin (SLN) is an integral membrane protein that is expressed in both skeletal and cardiac muscle, where it inhibits SERCA (calcium ATPase) by lowering its apparent Ca2+ affinity in a manner similar to that of its homologue phospholamban (PLN). We use solution NMR to map the structural changes occurring within SLN upon interaction with the regulatory target, SERCA, co-reconstituting the two proteins in dodecylphosphocholine (DPC) detergent micelles, a system that preserves the native structure of SLN and the activity of SERCA, with the goal of comparing these interactions with those of the previously studied PLN-SERCA complex. Our analysis of the structural dynamics of SLN in DPC micelles shows this polypeptide to be partitioned into four subdomains: a short unstructured N terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C terminus (residues 27-31). Upon addition of SERCA, the different domains behave according to their dynamics, molding onto the surface of the enzyme. Remarkably, each domain of SLN behaves in a manner similar to that of the corresponding domains in PLN, supporting the hypothesis that both SLN and PLN bind SERCA in the same groove and with similar mechanisms.

  13. Magnitude of finite-nucleus-size effects in relativistic density functional computations of indirect NMR nuclear spin-spin coupling constants.

    PubMed

    Autschbach, Jochen

    2009-09-14

    A spherical Gaussian nuclear charge distribution model has been implemented for spin-free (scalar) and two-component (spin-orbit) relativistic density functional calculations of indirect NMR nuclear spin-spin coupling (J-coupling) constants. The finite nuclear volume effects on the hyperfine integrals are quite pronounced and as a consequence they noticeably alter coupling constants involving heavy NMR nuclei such as W, Pt, Hg, Tl, and Pb. Typically, the isotropic J-couplings are reduced in magnitude by about 10 to 15 % for couplings between one of the heaviest NMR nuclei and a light atomic ligand, and even more so for couplings between two heavy atoms. For a subset of the systems studied, viz. the Hg atom, Hg(2) (2+), and Tl--X where X=Br, I, the basis set convergence of the hyperfine integrals and the coupling constants was monitored. For the Hg atom, numerical and basis set calculations of the electron density and the 1s and 6s orbital hyperfine integrals are directly compared. The coupling anisotropies of TlBr and TlI increase by about 2 % due to finite-nucleus effects.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26371375

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

    PubMed

    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. PMID:26371375

  17. Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T sub 1 NMR relaxation

    SciTech Connect

    Fraenkel, Y.; Navon, G. ); Aronheim, A.; Gershoni, J.M. )

    1990-03-13

    Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence {alpha}184-200 from the Torpedo californica receptor. This construct binds {alpha}-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control. In this study agonist binding to {alpha}184-200 is demonstrated by monitoring the T{sub 1} relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with {alpha}-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified {alpha}184-200 constructs, respectively. The K{sub D} values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while K{sub D}'s with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain {alpha}184-200 may comprise a major component of the cholinergic agonist site.

  18. The HSP90 binding mode of a radicicol-like E-oxime from docking, binding free energy estimations, and NMR 15N chemical shifts

    PubMed Central

    Spichty, Martin; Taly, Antoine; Hagn, Franz; Kessler, Horst; Barluenga, Sofia; Winssinger, Nicolas; Karplus, Martin

    2009-01-01

    We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of back-bone 15N provides an additional evaluation criteria. As a last test we check the binding modes against available structure-activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex. PMID:19482409

  19. Counter-ion binding and mobility in the presence of hydrophobic polyions – combining molecular dynamics simulations and NMR

    NASA Astrophysics Data System (ADS)

    Druchok, Maksym; Malikova, Natalie; Rollet, Anne-Laure; Vlachy, Vojko

    2016-06-01

    Counter-ion binding and mobility in aqueous solutions of partially hydrophobic ionene oligoions is studied here by a combination of all-atomic molecular dynamics (MD) simulations and NMR (19F and 81Br nuclei) measurements. We present results for 12, 12-ionenes in the presence of different halide ions (F-, Cl-, Br- and I-), as well as their mixtures; the latter allowing us to probe counter-ion selectivity of these oligoions. We consolidate both structural and dynamic information, in particular simulated radial distribution functions and average residence times of counter-ions in the vicinity of ionenes and NMR data in the form of counter-ion chemical shift and self-diffusion coefficients. On one hand, previously reported enthalpy of dilution and mixing measurements show a reverse counter-ion sequence for 12, 12-ionenes with respect to their less hydrophobic 3, 3- and 6, 6- analogues. On the other hand, the current MD and NMR data, reflecting the counter-ion binding tendencies to the ionene chain, give evidence for the same ordering as that observed by MD for 3, 3-ionenes. This is not seen as a contradiction and can be rationalized on the basis of increasing chain hydrophobicity, which has different consequences for enthalpy and ion-binding. The latter is reflecting free energy changes and as such includes both enthalpic and entropic contributions.

  20. Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy.

    PubMed

    Maiti, Mohitosh; Nauwelaerts, Koen; Lescrinier, Eveline; Herdewijn, Piet

    2011-02-01

    By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.

  1. Comparing Binding Modes of Analogous Fragments Using NMR in Fragment-Based Drug Design: Application to PRDX5

    PubMed Central

    Guichou, Jean-François; Cala, Olivier; Krimm, Isabelle

    2014-01-01

    Fragment-based drug design is one of the most promising approaches for discovering novel and potent inhibitors against therapeutic targets. The first step of the process consists of identifying fragments that bind the protein target. The determination of the fragment binding mode plays a major role in the selection of the fragment hits that will be processed into drug-like compounds. Comparing the binding modes of analogous fragments is a critical task, not only to identify specific interactions between the protein target and the fragment, but also to verify whether the binding mode is conserved or differs according to the fragment modification. While X-ray crystallography is the technique of choice, NMR methods are helpful when this fails. We show here how the ligand-observed saturation transfer difference (STD) experiment and the protein-observed 15N-HSQC experiment, two popular NMR screening experiments, can be used to compare the binding modes of analogous fragments. We discuss the application and limitations of these approaches based on STD-epitope mapping, chemical shift perturbation (CSP) calculation and comparative CSP sign analysis, using the human peroxiredoxin 5 as a protein model. PMID:25025339

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

    PubMed

    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

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

    PubMed

    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

  4. Cyclodextrins in pharmaceutical formulations II: solubilization, binding constant, and complexation efficiency.

    PubMed

    Jambhekar, Sunil S; Breen, Philip

    2016-02-01

    Cyclodextrins are cyclic oligosaccharides that have been recognized as pharmaceutical adjuvants for the past 20 years. The molecular structure of these glucose derivatives, which approximates a truncated cone, bucket, or torus, generates a hydrophilic exterior surface and a nonpolar interior cavity. Cyclodextrins can interact with appropriately sized drug molecules to yield an inclusion complex. These noncovalent inclusion complexes offer a variety of advantages over noncomplexed forms of a drug. Cyclodextrins are carbohydrates that are primarily used to enhance the aqueous solubility, physical chemical stability, and bioavailability of drugs. Their other applications include preventing drug-drug interactions, converting liquid drugs into microcrystalline powders, minimizing gastrointestinal and ocular irritation, and reducing or eliminating unpleasant taste and smell. Here, we focus on the solubilization of drugs by complexation, and discuss the determination and significance of binding constants for cyclodextrin complexes, and the determination of complexation efficiency and factors that influence it. We also make some general observations on cyclodextrin complexation and the use of cyclodextrins in solid, as well as parenteral, dosage forms. PMID:26687191

  5. Crystallographic and NMR evaluation of the impact of peptide binding to the second PDZ domain of PTP1E†

    PubMed Central

    Zhang, Jun; Sapienza, Paul J.; Ke, Hengming; Chang, Aram; Hengel, Sarah R.; Wang, Huanchen; Phillips, George N.; Lee, Andrew L.

    2010-01-01

    PDZ (PSD95/Discs large/ZO-1) domains are ubiquitous protein interaction motifs found in scaffolding proteins involved in signal transduction. Despite the fact that many PDZs show a limited tendency to undergo structural change, the PDZ family has been associated with long-range communication and allostery. One of the PDZ domains studied most in terms of structure and biophysical properties is the second PDZ (“PDZ2”) domain from protein tyrosine phophatase 1E (PTP1E, also known as PTPL1). Previously we showed through NMR relaxation studies that binding of the RA-GEF2 C-terminal peptide substrate results in long-range propagation of side-chain dynamic changes in human PDZ2 [Fuentes, et al., J. Mol. Biol. (2004), 335, 1105-1115]. Here, we present the first X-ray crystal structures of PDZ2 in the absence and presence of RA-GEF2 ligand, solved to resolutions of 1.65 and 1.3 Å, respectively. These structures deviate somewhat from previously determined NMR structures, and indicate that very minor structural changes in PDZ2 accompany peptide binding. NMR residual dipolar couplings confirm the crystal structures to be accurate models of the time-averaged atomic coordinates of PDZ2. The impact on side-chain dynamics was further tested with a C-terminal peptide from APC, which showed near-identical results to that of RA-GEF2. Thus, allosteric transmission in PDZ2 induced by peptide binding is conveyed purely and robustly by dynamics. 15N relaxation dispersion measurements did not detect appreciable populations of a kinetic structural intermediate. Collectively, for ligand binding to PDZ2, these data support a lock-and-key binding model from a structural perspective and an allosteric model from a dynamical perspective, which together suggest a complex energy landscape for functional transitions within the ensemble. PMID:20839809

  6. A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR.

    PubMed

    Sgourakis, Nikolaos G; May, Nathan A; Boyd, Lisa F; Ying, Jinfa; Bax, Ad; Margulies, David H

    2015-11-27

    As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent Ig-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full-length and truncated forms of the MHC-I molecule H2-L(d) by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-L(d), "mini-H2-L(d)," consisting of only the α1α2 platform domain. Mini-H2-L(d) refolded in vitro with a high affinity peptide yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the β2-microglobulin interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and β2-microglobulin-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the picosecond-nanosecond dynamics of the free mini-H2-L(d) MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway. PMID:26463211

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

    PubMed

    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.

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

    PubMed

    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. PMID:23464626

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

    PubMed

    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. 15N NMR investigation of the reduction and binding of TNT in an aerobic bench scale reactor simulating windrow composting

    USGS Publications Warehouse

    Thorn, K.A.; Pennington, J.C.; Hayes, C.A.

    2002-01-01

    T15NT was added to a soil of low organic carbon content and composted for 20 days in an aerobic bench scale reactor. The finished whole compost and fulvic acid, humic acid, humin, and lignocellulose fractions extracted from the compost were analyzed by solid-state CP/MAS and DP/MAS 15N NMR. 15N NMR spectra provided direct spectroscopic evidence for reduction of TNT followed by covalent binding of the reduced metabolites to organic matter of the composted soil, with the majority of metabolite found in the lignocellulose fraction, by mass also the major fraction of the compost. In general, the types of bonds formed between soil organic matter and reduced TNT amines in controlled laboratory reactions were observed in the spectra of the whole compost and fractions, confirming that during composting TNT is reduced to amines that form covalent bonds with organic matter through aminohydroquinone, aminoquinone, heterocyclic, and imine linkages, among others. Concentrations of imine nitrogens in the compost spectra suggestthat covalent binding bythe diamines 2,4DANT and 2,6DANT is a significant process in the transformation of TNT into bound residues. Liquid-phase 15N NMR spectra of the fulvic acid and humin fractions provided possible evidence for involvement of phenoloxidase enzymes in covalent bond formation.

  11. NMR and X-ray analysis of structural additivity in metal binding site-swapped hybrids of rubredoxin

    PubMed Central

    LeMaster, David M; Anderson, Janet S; Wang, Limin; Guo, Yi; Li, Hongmin; Hernández, Griselda

    2007-01-01

    Background Chimeric hybrids derived from the rubredoxins of Pyrococcus furiosus (Pf) and Clostridium pasteurianum (Cp) provide a robust system for the characterization of protein conformational stability and dynamics in a differential mode. Interchange of the seven nonconserved residues of the metal binding site between the Pf and Cp rubredoxins yields a complementary pair of hybrids, for which the sum of the thermodynamic stabilities is equal to the sum for the parental proteins. Furthermore, the increase in amide hydrogen exchange rates for the hyperthermophile-derived metal binding site hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid that is derived from the less thermostable rubredoxin, indicating a degree of additivity in the conformational fluctuations that underlie these exchange reactions. Results Initial NMR studies indicated that the structures of the two complementary hybrids closely resemble "cut-and-paste" models derived from the parental Pf and Cp rubredoxins. This protein system offers a robust opportunity to characterize differences in solution structure, permitting the quantitative NMR chemical shift and NOE peak intensity data to be analyzed without recourse to the conventional conversion of experimental NOE peak intensities into distance restraints. The intensities for 1573 of the 1652 well-resolved NOE crosspeaks from the hybrid rubredoxins were statistically indistinguishable from the intensities of the corresponding parental crosspeaks, to within the baseplane noise level of these high sensitivity data sets. The differences in intensity for the remaining 79 NOE crosspeaks were directly ascribable to localized dynamical processes. Subsequent X-ray analysis of the metal binding site-swapped hybrids, to resolution limits of 0.79 Å and 1.04 Å, demonstrated that the backbone and sidechain heavy atoms in the NMR-derived structures lie within the range of structural variability exhibited among the individual

  12. Quantification of ammonia binding sites in Davison (Type 3A) zeolite desiccant : a solid-state Nitrogen-15 MAS NMR spectroscopy investigation.

    SciTech Connect

    Alam, Todd Michael; Holland, Gregory P.; Cherry, Brian Ray

    2004-01-01

    The quantitative analysis of ammonia binding sites in the Davison (Type 3A) zeolite desiccant using solid-state {sup 15}N MAS NMR spectroscopy is reported. By utilizing 15N enriched ammonia ({sup 15}NH{sub 3}) gas, the different adsorption/binding sites within the zeolite were investigated as a function of NH{sub 3} loading. Using {sup 15}N MAS NMR multiple sites were resolved that have distinct cross-polarization dynamics and chemical shift behavior. These differences in the {sup 15}N NMR were used to characterize the adsorption environments in both the pure 3A zeolite and the silicone-molded forms of the desiccant.

  13. NMR structure of a biologically active peptide containing the RNA-binding domain of human immunodeficiency virus type 1 Tat.

    PubMed Central

    Mujeeb, A; Bishop, K; Peterlin, B M; Turck, C; Parslow, T G; James, T L

    1994-01-01

    The Tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific RNA element on nascent viral transcripts. Binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. Here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 Tat basic domain linked to the core regulatory domain of another lentiviral Tat--i.e., that from equine infectious anemia virus. Circular dichroism and two-dimensional proton NMR studies of this hybrid peptide indicate that the Tat basic domain forms a stable alpha-helix, whereas the adjacent regulatory sequence is mostly in extended form. These findings suggest that the tendency to form stable alpha-helices may be a common property of arginine- and lysine-rich RNA-binding domains. Images PMID:8058789

  14. Analysis of phi and chi 1 torsion angles for hen lysozyme in solution from 1H NMR spin-spin coupling constants.

    PubMed

    Smith, L J; Sutcliffe, M J; Redfield, C; Dobson, C M

    1991-01-29

    Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J alpha beta coupling constants have been measured for 57 residues of the 129-residue protein hen egg white lysozyme. These NMR data have been compared with torsion angles defined in the tetragonal and the triclinic crystal forms of the protein. For most residues the measured 3JHN alpha values were consistent with the phi torsion angles found in both crystal forms; the RMS difference between the coupling constants calculated by using the tetragonal crystal structure phi angles and the experimental 3JHN alpha values is 0.88 Hz. Thus there appears to be no significant averaging of the phi torsion angle either in the interior or at the surface of the protein. For 41 of the residues where 3J alpha beta coupling constants have been determined, the values are consistent with a single staggered conformation about the chi 1 torsion angle and there is complete agreement between the NMR data in solution and the torsion angles defined in the crystalline state. In contrast, for the other 16 residues where 3J alpha beta coupling constant values have been measured, the data indicate extensive motional averaging about the chi 1 torsion angle. These residues occur largely on the surface of the protein and examination of the crystal structures shows that many of these residues adopt a different conformation in the triclinic and tetragonal crystal forms and have high crystallographic temperature factors. It appears, however, that in solution conformational flexibility of the side chains of surface residues is significantly more pronounced than in individual crystal structures.

  15. Structural Requirements for Bisphosphonate Binding on Hydroxyapatite: NMR Study of Bisphosphonate Partial Esters

    PubMed Central

    2015-01-01

    Eighteen different bisphosphonates, including four clinically used bisphosphonate acids and their phosphoesters, were studied to evaluate how the bisphosphonate structure affects binding to bone. Bisphosphonates with weak bone affinity, such as clodronate, could not bind to hydroxyapatite after the addition of one ester group. Medronate retained its ability to bind after the addition of one ester group, and hydroxy-bisphosphonates could bind even after the addition of two ester groups. Thus, several bisphosphonate esters are clearly bone binding compounds. The following conclusions about bisphosphonate binding emerge: (1) a hydroxyl group in the geminal carbon takes part in the binding process and increases the bisphosphonate’s ability to bind to bone; (2) the bisphosphonate’s ability to bind decreases when the amount of ester groups increases; and (3) the location of the ester groups affects the bisphosphonate’s binding ability. PMID:25893039

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

    PubMed

    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. PMID:20178991

  17. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein.

  18. 15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid, model compounds, and lignocellulose

    USGS Publications Warehouse

    Thorn, K.A.; Kennedy, K.R.

    2002-01-01

    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.

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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. PMID:26426398

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

    NASA Astrophysics Data System (ADS)

    Antušek, Andrej; Keḑziera, Dariusz; Jackowski, Karol; Jaszuński, Michał; Makulski, Włodzimierz

    2008-09-01

    New values of the indirect spin-spin coupling constants in CH 4, SiH 4 and GeH 4, derived from experiment and ab initio calculations, are reported. The new experimental values of 1J(CH), 1J(SiH) and 1J(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 1J(XH), X = C, Si, Ge, constants is very satisfying, the differences are approximately 1-3%.

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

    SciTech Connect

    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

  5. What determines the sign of the Fermi-contact contribution to the NMR spin spin coupling constant?

    NASA Astrophysics Data System (ADS)

    Del Bene, Janet E.; Elguero, José

    2003-11-01

    The Dirac vector model for the sign of the spin-spin coupling constant ( J) between a pair of atoms states that one-bond coupling constants are positive, two-bond negative, three-bond positive, etc. However, this rule is often violated. In an attempt to understand the sign of J, we propose a new model, the Nuclear magnetic resonance triplet wavefunction model (NMRTWM) which is based on the phases of excited triplet state wavefunctions, and the response of magnetic nuclei to these phases. The model demonstrates that, (1) the excited states which contribute to the coupling constant do so in a regular manner; (2) the sign of an individual contribution is determined by the nodal character of the excited-state wavefunction and the response of the nuclei to the phases of this function; and (3) the sign of the total coupling constant is the result of competing positive and negative contributions from various states. NMRTWM provides a fundamental explanation for both the successes and failures of the Dirac vector model, and can be used to gain insight into some puzzling results for the orientation dependence of F-F coupling between two HF molecules.

  6. Competitive counterion complexation allows the true host : guest binding constants from a single titration by ionic receptors.

    PubMed

    Pessêgo, Márcia; Basílio, Nuno; Muñiz, M Carmen; García-Río, Luis

    2016-07-01

    Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration.

  7. Competitive counterion complexation allows the true host : guest binding constants from a single titration by ionic receptors.

    PubMed

    Pessêgo, Márcia; Basílio, Nuno; Muñiz, M Carmen; García-Río, Luis

    2016-07-01

    Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration. PMID:27278457

  8. Solution secondary structure of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK: A heteronuclear multidimensional NMR study.

    PubMed

    Campbell, A P; Bautista, D L; Tripet, B; Wong, W Y; Irvin, R T; Hodges, R S; Sykes, B D

    1997-10-21

    The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has recently been the target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. We have successfully cloned and bacterially expressed a 15N-labeled PAK pilin peptide spanning residues 128-144 of the intact PAK pilin protein, PAK 128-144(Hs145), and have determined the solution secondary structure of this peptide using heteronuclear multidimensional NMR spectroscopy. The oxidized recombinant peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around the Ile138-Pro139 peptide bond. The pattern of NOEs, temperature coefficients, and coupling constants observed for the trans isomer demonstrate the presence of a type I beta-turn and a type II beta-turn spanning Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively. This is in agreement with the NMR solution structure of the trans isomer of a synthetic PAK 128-144 peptide which showed a type I and a type II beta-turn in these same regions of the sequence [McInnes, C., Sönnichsen, F. D., Kay, C. M., Hodges, R. S., and Sykes, B. D. (1993) Biochemistry 32, 13432-13440; Campbell, A. P., McInnes, C., Hodges, R. S., and Sykes, B. D. (1995) Biochemistry 34, 16255-16268]. The pattern of NOEs, temperature coefficients, and coupling constants observed for the cis isomer also demonstrate a type II beta-turn spanning Pro139-Lys-Gly-Cys142, but suggest a second beta-turn spanning Asp132-Gln-Asp-Glu135. Thus, the cis isomer may also possess a double-turn motif (like the trans isomer), but with different spacing between the turns and a different placement of the first turn in the sequence. The discovery of a double-turn motif in the trans (and cis) recombinant PAK pilin peptide is an extremely important result since the double turn has been implicated as a structural requirement for the recognition of both receptor

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

    PubMed

    Meneses, Erick; Mittermaier, Anthony

    2014-10-01

    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.

  10. NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

    PubMed Central

    Thammaporn, Ratsupa; Yagi-Utsumi, Maho; Yamaguchi, Takumi; Boonsri, Pornthip; Saparpakorn, Patchreenart; Choowongkomon, Kiattawee; Techasakul, Supanna; Kato, Koichi; Hannongbua, Supa

    2015-01-01

    Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs. PMID:26510386

  11. Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

    PubMed

    Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points.

  12. The Binding Constant of Estradiol to Bovine Serum Albumin: An Upper-Level Experiment Utilizing Tritium-Labeled Estradiol and Liquid Scintillation Counting

    ERIC Educational Resources Information Center

    Peihong Liang; Adhyaru, Bhavin; Pearson, Wright L.; Williams, Kathryn R.

    2006-01-01

    The experiment used [to the third power]H-labeled estradiol to determine the binding constant of estradiol to bovine serum albumin. Estradiol must complex with serum proteins for the transport in the blood stream because of its low solubility in aqueous systems and estradiol-protein binding constant, where K[subscript B] is important to understand…

  13. Non-empirical calculations of NMR indirect carbon-carbon coupling constants. Part 9--Bicyclobutane-containing polycycloalkanes.

    PubMed

    Krivdin, Leonid B

    2004-10-01

    13C--(13)C spin-spin coupling constants characterizing the bicyclobutane moiety of seven well-known bicyclobutane-containing polycycloalkanes were calculated at the SOPPA level. Benchmark calculations on tricyclopentane and octabisvalene revealed an appropriate level of theory and sufficient quality of basis sets used to perform geometry searches and to calculate spin-spin coupling constants. Several experimental uncertainties were resolved and a number of interesting couplings were predicted. The most interesting trend observed in this series of polycycloalkanes is the marked increase (decrease in absolute value) of J(C,C) between bridgehead carbons with increase in the puckering angle of the bicyclobutane moiety. This predicts almost zero coupling between bridgehead carbons of tricyclopentane and explains the positive J(C,C) in tetrahedrane in contrast to the negative bridgehead-bridgehead J(C,C) in bicyclobutane.

  14. Determination of the binding constant of indomethacin-beta-cyclodextrin complex by capillary electrophoresis: experimental optimization and temperature study.

    PubMed

    Acosta, G; Linares, D; Olsina, R; Martínez, L D; Gomez, M R

    2007-11-01

    The apparent electrophoretic mobilities of indomethacin in beta-cyclodextrin at a range of concentrations were measured directly by capillary electrophoresis. Three different linear plots and a non linear plot are proposed for the apparent binding constant calculations, based on the fact that the molar ratio of the inclusion complex was 1:1. K values obtained at 298 K were 421 M(-1) (double reciprocal fit), 488 M(-1) (x-reciprocal fit), 428 M(-1) (y-reciprocal fit) and 490 M(-1) (non linear fit). The corresponding K values at 313 K were 380 M(-1) (double reciprocal fit), 355 M(-1) (x-reciprocal fit), 366 M(-1) (y-reciprocal fit) and 339 M(-1) (non linear fit). Using the proposed methods, the binding constant of the indomethacin-beta-cyclodextrin inclusion complex can be obtained easily. The methods have been applied to obtain the values of the constant K under different experimental conditions. Under optimized conditions the K constant is temperature dependent and non-arrhenian behaviour was observed.

  15. Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

    The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ( 1H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sułkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

  16. NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

    PubMed Central

    Aitio, H.; Annila, A.; Heikkinen, S.; Thulin, E.; Drakenberg, T.; Kilpeläinen, I.

    1999-01-01

    Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum. PMID:10631973

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

    PubMed

    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.

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

    PubMed

    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. PMID:25885091

  19. (19)F NMR studies of the leucine-isoleucine-valine binding protein: evidence that a closed conformation exists in solution.

    PubMed

    Salopek-Sondi, Branka; Vaughan, Mark D; Skeels, Matthew C; Honek, John F; Luck, Linda A

    2003-10-01

    The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This "venus fly trap" type protein has been characterized by crystallography in only the open form. Herein we have labeled LIV with 5-fluorotryptophan (5F-Trp) and difluoromethionine (DFM) in order to explore the structural dynamics of this protein and the application of DFM as a potential (19)F NMR structural probe for this family of proteins. Based on mass spectrometric analysis of the protein overproduced in the presence of DFM, approximately 30% of the five LIV methionine residues were randomly substituted with the fluorinated analog. Urea denaturation experiments imply a slight decrease in protein stability when DFM is incorporated into LIV. However, the fluorinated methionine did not alter leucine-binding activity upon its incorporation into the protein. Binding of L-leucine stabilizes both the unlabeled and DFM-labeled LIV, and induces the protein to adopt a three-state unfolding model in place of the two-state process observed for the free protein. The (19)F NMR spectrum of DFM-labeled LIV gave distinct resonances for the five Met residues found in LIV. 5F-Trp labeled LIV gave a well resolved spectrum for the three Trp residues. Trp to Phe mutants defined the resonances in the spectrum. The distinct narrowing in line width of the resonances when ligand was added identified the closed form of the protein. PMID:12956607

  20. Revealing the specific solute-solvent interactions via the measurements of the NMR spin-spin coupling constants

    NASA Astrophysics Data System (ADS)

    Shahkhatuni, Astghik A.; Shahkhatuni, Aleksan G.; Minasyan, Nune S.; Panosyan, Henry A.; Sahakyan, Aleksandr B.

    2015-03-01

    The solvent induced changes of one-bond spin-spin coupling constants (SSCCs) are investigated for a set of substituted methanes in solvents with various ε dielectric constants. Solute-solvent systems with varying types of ε-dependences for the solute SSCCs are outlined. Aliphatic hydrocarbon solvents and their halogen-substituted derivatives comprise the subset, where the SSCC is linearly dependent on the solvent reaction field, f(ε) = 2(ε - 1)/(2ε + 1), hence indicating the absence of specific solute-solvent interactions. In such solvents, SSCCs depend only on bulk dielectric properties of the medium, and, the magnitudes of the solvent sensitivities of SSCCs are fully determined by the initial values of "pure" SSCCs that correspond to the isolated solute molecules. The solvents involved in the second subset have a relatively chaotic distribution of the SSCC/f(ε) relationship, with possible groupings by their chemical nature. There, the conventional linear SSCC/f(ε) dependence is perturbed by additional interactions, such as hydrogen bonding, specific association processes, lone electron pairs, and conjugation.

  1. Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.

    PubMed

    Park, Seyeon; Chung, Sunah; Kim, Kyung-Mee; Jung, Kyung-Chae; Park, Chihoon; Hahm, Eun-Ryeong; Yang, Chul-Hak

    2004-02-24

    The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation. The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively. When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over

  2. The binding of metal ions and angiotensin converting enzyme (ACE) inhibitor by 13C NMR

    NASA Astrophysics Data System (ADS)

    Sakamoto, Yohko; Sakamoto, Yuko; Ishii, Tomoko; Ohmoto, Taichi

    1991-06-01

    Enalaprilat (MK-422, 1- [ N- [1 (S)-carboxy-3-phenylpropyl]- L-alanyl]- L-proline (1)) and Lisinopril (MK521, N- N- [ (s)-l-carboxy-3- phenylpropyl]- L-lysyl- L-proline, (2)) exhibit the capacity to act as a chelate, unidentate or bridge towards metal ions in aqueous solution, as determined by 13C NMR. By adding metal ions, in the series of Zn 2+, Ni 2+, Pb 2+, Pd 2+ and Cd 2+, the active site of the ACE inhibitor was well defined. MK-521 was more influenced by nuclei that were distant from the active site than MK-422.

  3. NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

    PubMed Central

    Alag, Reema; Qureshi, Insaf A; Bharatham, Nagakumar; Shin, Joon; Lescar, Julien; Yoon, Ho Sup

    2010-01-01

    The emergence of drug-resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X-ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites. PMID:20572013

  4. The RXR{alpha} C-terminus T462 is a NMR sensor for coactivator peptide binding

    SciTech Connect

    Lu Jianyun Chen Minghe; DeKoster, Gregory T.; Cistola, David P.; Li, Ellen

    2008-02-22

    The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXR{alpha})-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXR{alpha} ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K{sub d} derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the {sup 15}N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.

  5. Spectrophotometric Determination of Iron(III)-Glycine Formation Constant in Aqueous Medium Using Competitive Ligand Binding

    ERIC Educational Resources Information Center

    Prasad, Rajendra; Prasad, Surendra

    2009-01-01

    The formation constant of iron(III) complex with glycine (Gly) ligand in aqueous acidic medium (0.2 M HNO[subscript 3], I = 0.2 M at 28 plus or minus 1 degree C) was determined spectrophotometrically in which a competing color reaction between Fe(III) and SCN[superscript -] was used as an indicator reaction. Under the specified conditions Fe(III)…

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

    PubMed

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

    2012-03-01

    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).

  7. NMR spin-spin coupling constants: bond angle dependence of the sign and magnitude of the vicinal (3)JHF coupling.

    PubMed

    Viesser, Renan V; Ducati, Lucas C; Autschbach, Jochen; Tormena, Cláudio F

    2016-08-24

    The dependence of the magnitude and sign of (3)JHFF on the bond angle in fluoro-cycloalkene compounds is evaluated by electronic structure calculations using different levels of theory, viz. DFT, SOPPA(CCSD) and SOPPA(CC2). Localized molecular orbital contributions to (3)JHFF are analyzed to assess which orbitals are responsible for (3)JHFF and which are the most important coupling transmission mechanisms for each compound. Fluoro-ethylene is used as a model system to evaluate the dependence of the (3)JHFF coupling constant on the angle between the σCα-F and σCα'-HF vectors. Through-space and hyperconjugative transmission pathways and ring strain are identified as responsible for the opposite trend between (3)JHFF and bond angle, and for the negative signs obtained for the two molecules, respectively. One of the fluorine lone pairs, σCα'-HF, σCα-F, σCα'-Cβ' bonding orbitals and the σ*Cα-F antibonding orbital are involved in the J-coupling pathways, according to analyses of pairwise-steric and hyperconjugative energies. PMID:27526856

  8. NMR spin-spin coupling constants: bond angle dependence of the sign and magnitude of the vicinal (3)JHF coupling.

    PubMed

    Viesser, Renan V; Ducati, Lucas C; Autschbach, Jochen; Tormena, Cláudio F

    2016-08-24

    The dependence of the magnitude and sign of (3)JHFF on the bond angle in fluoro-cycloalkene compounds is evaluated by electronic structure calculations using different levels of theory, viz. DFT, SOPPA(CCSD) and SOPPA(CC2). Localized molecular orbital contributions to (3)JHFF are analyzed to assess which orbitals are responsible for (3)JHFF and which are the most important coupling transmission mechanisms for each compound. Fluoro-ethylene is used as a model system to evaluate the dependence of the (3)JHFF coupling constant on the angle between the σCα-F and σCα'-HF vectors. Through-space and hyperconjugative transmission pathways and ring strain are identified as responsible for the opposite trend between (3)JHFF and bond angle, and for the negative signs obtained for the two molecules, respectively. One of the fluorine lone pairs, σCα'-HF, σCα-F, σCα'-Cβ' bonding orbitals and the σ*Cα-F antibonding orbital are involved in the J-coupling pathways, according to analyses of pairwise-steric and hyperconjugative energies.

  9. Large structure rearrangement of colicin ia channel domain after membrane binding from 2D 13C spin diffusion NMR.

    PubMed

    Luo, Wenbin; Yao, Xiaolan; Hong, Mei

    2005-05-01

    One of the main mechanisms of membrane protein folding is by spontaneous insertion into the lipid bilayer from the aqueous environment. The bacterial toxin, colicin Ia, is one such protein. To shed light on the conformational changes involved in this dramatic transfer from the polar to the hydrophobic milieu, we carried out 2D magic-angle spinning (13)C NMR experiments on the water-soluble and membrane-bound states of the channel-forming domain of colicin Ia. Proton-driven (13)C spin diffusion spectra of selectively (13)C-labeled protein show unequivocal attenuation of cross-peaks after membrane binding. This attenuation can be assigned to distance increases but not reduction of the diffusion coefficient. Analysis of the statistics of the interhelical and intrahelical (13)C-(13)C distances in the soluble protein structure indicates that the observed cross-peak reduction is well correlated with a high percentage of short interhelical contacts in the soluble protein. This suggests that colicin Ia channel domain becomes open and extended upon membrane binding, thus lengthening interhelical distances. In comparison, cross-peaks with similar intensities between the two states are dominated by intrahelical contacts in the soluble state. This suggests that the membrane-bound structure of colicin Ia channel domain may be described as a "molten globule", in which the helical secondary structure is retained while the tertiary structure is unfolded. This study demonstrates that (13)C spin diffusion NMR is a valuable tool for obtaining qualitative long-range distance constraints on membrane protein folding. PMID:15853348

  10. Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants.

    PubMed

    Shapiro, Adam B; Gao, Ning; Gu, Rong-Fang; Thresher, Jason

    2014-10-15

    High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37-43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.

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

    PubMed

    Gervais, Virginie; Campagne, Sébastien; Durand, Jade; Muller, Isabelle; Milon, Alain

    2013-05-01

    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. PMID:23306615

  12. Analysis of the binding of high mobility group protein 17 to the nucleosome core particle by 1H NMR spectroscopy.

    PubMed

    Cook, G R; Minch, M; Schroth, G P; Bradbury, E M

    1989-01-25

    The binding of high mobility group (HMG) protein 17 to the nucleosome core particle has been studied in D2O solution using 1H NMR at 500 MHz. Spectra were obtained for purified HMG 17, purified nucleosome core particles, and the reconstituted HMG 17-nucleosome core particle complex at 0.1, 0.2, 0.3, and 0.4 M NaCl. Subtraction of the core particle spectra from spectra of the core particle reconstituted with HMG 17 demonstrated those regions of HMG 17 which interact with the nucleosome at different ionic strengths; the resonance peaks of interacting groups are broadened due to their restricted mobility. At 0.1 M NaCl, the mobility of all the amino acid side chains of HMG 17 was restricted, indicating complete binding of HMG 17 to the much larger nucleosome core particle. At 0.2 M NaCl most of the amino acids were free with the exception of arginine and proline which are confined to or predominant in the basic central region of HMG 17. These amino acids were completely free only at 0.4 M NaCl. We conclude that the entire HMG 17 molecule interacts with the nucleosome core particle at physiological ionic strength. The acidic COOH-terminal region of HMG 17 is released from interaction with the core histones at an NaCl concentration between 0.1 and 0.2 M and so binds weakly at physiological ionic strength. The basic central region binds more strongly to the core particle DNA, being completely released only at much higher ionic strength, between 0.3 and 0.4 M NaCl.

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

    EPA Science Inventory

    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

  14. NMR structure and binding of esculentin-1a (1-21)NH2 and its diastereomer to lipopolysaccharide: Correlation with biological functions.

    PubMed

    Ghosh, Anirban; Bera, Swapna; Shai, Yechiel; Mangoni, Maria Luisa; Bhunia, Anirban

    2016-04-01

    The frog skin-derived antimicrobial peptide esculentin-1a(1-21)NH2 [Esc(1-21)], and its diastereomer Esc(1-21)-1c (containing two D-amino acids at positions 14 and 17), have been recently found to neutralize the toxic effect of Pseudomonas aeruginosa lipopolysaccharide (LPS), although to different extents. Here, we studied the three-dimensional structure of both peptides in complex with P. aeruginosa LPS, by transferred nuclear Overhauser effect spectroscopy. Lack of NOE peaks revealed that both the peptides adopted a random coil structure in aqueous solution. However, Esc(1-21) adopted an amphipathic helical conformation in LPS micelles with 5 basic Lys residues forming a hydrophilic cluster. In comparison, the diastereomer maintained an alpha helical conformation only at the N-terminal region, whereas the C-terminal portion was quite flexible. Isothermal titration calorimetry (ITC) revealed that the interaction of Esc(1-21) with LPS is an exothermic process associated with a dissociation constant of -4μM. In contrast, Esc(1-21)-1c had almost 8 times weaker binding affinity to LPS micelles. Moreover, STD NMR data supported by docking analysis have identified those amino acid residues responsible for the peptide's binding to LPS micelles. Overall, the data provide important mechanistic insights on the interaction of esculentin-derived peptides with LPS and the reason for their different anti-endotoxin activity. These data might also assist to further design more potent antimicrobial peptides with antisepsis properties, which are highly needed to overcome the widespread concern of the available anti-infective agents. PMID:26724203

  15. NMR study of the phosphoryl binding loop in purine nucleotide proteins: Evidence for strong hydrogen binding in human N-ras p21

    SciTech Connect

    Redfield, A.G.; Papastavros, M.Z. )

    1990-04-10

    The structure of the phosphoryl binding region of human N-ras p21 was probed by using heteronuclear proton-observed NMR methods. Normal protein and a Gly-12 {yields} Asp-12 mutant protein were prepared with two amino acids labeled with {sup 15}N at their amide positions: valine and glycine, aspartic acid and glycine, and lysine and glycine. The authors completed the identification of amide {sup 15}NH resonances from Gly-12 and Asp-12 to the end of the phosphoryl binding domain consensus sequence (Lys-16) in protein complexed with GDP and have made tentative amide identifications from Val-9 to Ser-17. The methods used, together with initial identifications of the Gly-12 and -13 amide resonances, were described previously. The amide resonances of both Gly-13 and Lys-16 are shifted downfield below 10.4 ppm in both the normal and mutant proteins. These downfield shifts are presumed to be due to strong hydrogen bonds with the {beta}-phosphate of GDP.

  16. 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)

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    EPA Science Inventory

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

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

    NASA Astrophysics Data System (ADS)

    Zhang, Rongchun; Ramamoorthy, Ayyalusamy

    2016-01-01

    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 1H/13C/1H 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 13C/1H and 13C/13C chemical shift correlations, the 3D 1H/13C/1H experiment also provides a COSY-type 1H/1H 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 (1H/1H chemical shift correlation spectrum) at different 13C chemical shift frequencies from the 3D 1H/13C/1H 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 1H/13C/1H experiment would be useful to study the structure and dynamics of a variety of chemical and biological

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

    PubMed

    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

  1. Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments.

    PubMed

    Constantine, Keith L; Davis, Malcolm E; Metzler, William J; Mueller, Luciano; Claus, Brian L

    2006-06-01

    Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.

  2. 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.

    PubMed

    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.

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

    ERIC Educational Resources Information Center

    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…

  4. /sup 113/Cd NMR studies of a 1:1 Cd adduct with an 18-residue finger peptide from HIV-1 nucleic acid binding protein, p7

    SciTech Connect

    South, T.L.; Kim, B.; Summers, M.F.

    1989-01-04

    The Zn/sup 2+/ and Cd/sup 2+/ adducts with the 18-residue peptide comprising the amino acid sequence of the first finger (residues 13 through 30) of retroviral nucleic acid binding proteins p7 from HIV-1 (the causative agent of AIDS) have been prepared. /sup 1/H NMR data indicate that the metal adducts are 1:1 compounds that are stable in aqueous solutions for at least a month. The /sup 113/Cd NMR spectral results for the adduct are presented and analyzed. 26 references, 3 figures.

  5. NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites.

    PubMed

    Di Lello, Paola; Nguyen, Bao D; Jones, Tamara N; Potempa, Krzysztof; Kobor, Michael S; Legault, Pascale; Omichinski, James G

    2005-05-31

    General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEalpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1(1-115)). Like the corresponding domain from the human p62 protein, Tfb1(1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta5-beta7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation. PMID:15909982

  6. Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study.

    PubMed

    Krishna Sastry, M V; Swamy, M J; Surolia, A

    1988-10-15

    Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.

  7. Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

    PubMed

    Aramini, James M; Hamilton, Keith; Rossi, Paolo; Ertekin, Asli; Lee, Hsiau-Wei; Lemak, Alexander; Wang, Huang; Xiao, Rong; Acton, Thomas B; Everett, John K; Montelione, Gaetano T

    2012-05-01

    Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro.

  8. Solution NMR structure, backbone dynamics, and heme-binding properties of a novel cytochrome c maturation protein CcmE from Desulfovibrio vulgaris.

    PubMed

    Aramini, James M; Hamilton, Keith; Rossi, Paolo; Ertekin, Asli; Lee, Hsiau-Wei; Lemak, Alexander; Wang, Huang; Xiao, Rong; Acton, Thomas B; Everett, John K; Montelione, Gaetano T

    2012-05-01

    Cytochrome c maturation protein E, CcmE, plays an integral role in the transfer of heme to apocytochrome c in many prokaryotes and some mitochondria. A novel subclass featuring a heme-binding cysteine has been identified in archaea and some bacteria. Here we describe the solution NMR structure, backbone dynamics, and heme binding properties of the soluble C-terminal domain of Desulfovibrio vulgaris CcmE, dvCcmE'. The structure adopts a conserved β-barrel OB fold followed by an unstructured C-terminal tail encompassing the CxxxY heme-binding motif. Heme binding analyses of wild-type and mutant dvCcmE' demonstrate the absolute requirement of residue C127 for noncovalent heme binding in vitro. PMID:22497251

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

    PubMed

    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.

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

    PubMed

    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.

  11. Quantum-chemical simulation of 1H NMR spectra. 2. Comparison of DFT-based procedures for computing proton-proton coupling constants in organic molecules.

    PubMed

    Bally, Thomas; Rablen, Paul R

    2011-06-17

    The performance of 250 different computational protocols (combinations of density functionals, basis sets and methods) was assessed on a set of 165 well-established experimental (1)H-(1)H nuclear coupling constants (J(H-H)) from 65 molecules spanning a wide range of "chemical space". Thereby we found that, if one uses core-augmented basis sets and allows for linear scaling of the raw results, calculations of only the Fermi contact term yield more accurate predictions than calculations where all four terms that contribute to J(H-H) are evaluated. It turns out that B3LYP/6-31G(d,p)u+1s is the best (and, in addition, one of the most economical) of all tested methods, yielding predictions of J(H-H) with a root-mean-square deviation from experiment of less than 0.5 Hz for our test set. Another method that does similarly well, without the need for additional 1s basis functions, is B3LYP/cc-pVTZ, which is, however, ca. 8 times more "expensive" in terms of CPU time. A selection of the better methods was tested on a probe set comprising 61 J(H-H) values from 37 molecules. In this set we also included five molecules where conformational averaging is required. The rms deviations were better than or equal to those with the training set, which indicates that the method we recommend is generally applicable for organic molecules. We give instructions on how to carry out calculations of (1)H chemical shifts and J(H-H) most economically and provide scripts to extract the relevant information from the outputs of calculations with the Gaussian program in clearly arranged form, e.g., to feed them into programs for simulating entire (1)H NMR spectra.

  12. Assignment of the sup 1 H NMR spectrum and secondary structure elucidation of the single-stranded DNA binding protein encoded by the filamentous bacteriophage IKe

    SciTech Connect

    van Duynhoven, J.P.M.; Folkers, P.J.M.; Prinse, C.W.J.M.; Harmsen, B.J.M.; Konings, R.N.H.; Hilbers, C.W. )

    1992-02-04

    By means of 2D NMR techniques, all backbone resonances in the {sup 1}H NMR spectrum of the single-stranded DNA binding protein encoded by gene V of the filamentous phage IKe have been assigned sequence specifically. In addition, a major part of the side chain resonances could be assigned as well. Analysis of NOESY data permitted the elucidation of the secondary structure of IKe gene V protein. The major part of the secondary structure is present as an antiparallel {beta}-sheet, i.e., as two {beta}-loops which partly combine into a triple-stranded {beta}-sheet structure, one {beta}-loop and one triple-stranded {beta}-sheet structure. It is shown that a high degree of homology exists with the single-stranded DNA binding protein encoded by gene V of the distantly related filamentous phase M13.

  13. Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of {alpha}-synuclein

    SciTech Connect

    Sasakawa, Hiroaki |; Sakata, Eri; Yamaguchi, Yoshiki; Masuda, Masami |; Mori, Tetsuya; Kurimoto, Eiji; Iguchi, Takeshi; Hisanaga, Shin-ichi; Iwatsubo, Takeshi; Hasegawa, Masato; Kato, Koichi |

    2007-11-23

    Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of {alpha}-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-{alpha}-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of {alpha}-synuclein.

  14. Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR

    PubMed Central

    Marius, Phedra; de Planque, Maurits R.R.; Williamson, Philip T.F.

    2012-01-01

    The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channel's subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening. PMID:21963409

  15. 1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

    PubMed Central

    Feher, V. A.; Zapf, J. W.; Hoch, J. A.; Dahlquist, F. W.; Whiteley, J. M.; Cavanagh, J.

    1995-01-01

    Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54. PMID:8528078

  16. NMR-Assisted Molecular Docking Methodologies.

    PubMed

    Sturlese, Mattia; Bellanda, Massimo; Moro, Stefano

    2015-08-01

    Nuclear magnetic resonance (NMR) spectroscopy and molecular docking are regularly being employed as helpful tools of drug discovery research. Molecular docking is an extremely rapid method to evaluate possible binders from a large chemical library in a fast and cheap manner. NMR techniques can directly detect a protein-ligand interaction, can determine the corresponding association constant, and can consistently identify the ligand binding cavity. Consequently, molecular docking and NMR techniques are naturally complementary techniques where the combination of the two has the potential to improve the overall efficiency of drug discovery process. In this review, we would like to summarize the state of the art of docking methods which have been recently bridged to NMR experiments to identify novel and effective therapeutic drug candidates.

  17. NMR-Assisted Molecular Docking Methodologies.

    PubMed

    Sturlese, Mattia; Bellanda, Massimo; Moro, Stefano

    2015-08-01

    Nuclear magnetic resonance (NMR) spectroscopy and molecular docking are regularly being employed as helpful tools of drug discovery research. Molecular docking is an extremely rapid method to evaluate possible binders from a large chemical library in a fast and cheap manner. NMR techniques can directly detect a protein-ligand interaction, can determine the corresponding association constant, and can consistently identify the ligand binding cavity. Consequently, molecular docking and NMR techniques are naturally complementary techniques where the combination of the two has the potential to improve the overall efficiency of drug discovery process. In this review, we would like to summarize the state of the art of docking methods which have been recently bridged to NMR experiments to identify novel and effective therapeutic drug candidates. PMID:27490497

  18. Covalent binding of reduced metabolites of [{sup 15}N{sub 3}]TNT to soil organic matter during a bioremediation process analyzed by {sup 15}N NMR spectroscopy

    SciTech Connect

    Achtnich, C.; Fernandes, E.; Bollag, J.M.; Knackmuss, H.J.; Lenke, H.

    1999-12-15

    Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-{sup 15}N{sub 3}-trinitrotoluene (TNT) to different soil fractions, using liquid {sup 15}N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-{sup 15}N{sub 3}-trinitrotoluene and {sup 14}C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly whereas the radioactivity bound to humin increased to 71%. The {sup 15}N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, {sup 15}N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

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

    PubMed

    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. Using electrophoretic mobility shift assays to measure equilibrium dissociation constants: GAL4-p53 binding DNA as a model system.

    PubMed

    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(D) ) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel; the DNA migrates more slowly when bound to a protein. To determine a K(D) the amount of unbound and protein-bound DNA in the gel is measured as the protein concentration increases. By performing this experiment, students will be introduced to making affinity measurements and gain experience in performing quantitative EMSAs. The experiment describes measuring the K(D) for the interaction between the chimeric protein GAL4-p53 and its DNA recognition site; however, the techniques are adaptable to other DNA binding proteins. In addition, the basic experiment described can be easily expanded to include additional inquiry-driven experimentation. © 2012 by The International Union of Biochemistry and Molecular Biology.

  1. Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: Assignment of. beta. -sheet and flavin binding site resonances and analysis of protein-flavin interactions

    SciTech Connect

    Stockman, B.J.; Krezel, A.M.; Markley, J.L. ); Leonhardt, K.G.; Straus, N.A. )

    1990-10-01

    Sequence-specific {sup 1}H and {sup 13}C NMR assignments have been made for residues that form the five-stranded parallel {beta}-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the {beta}-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans. A total of 62 NOEs were identified: 8 between protons of bound FMN, 29 between protons of the protein in the flavin binding site, and 25 between protons of bound FMN and protons of the protein. These constraints were used to determine the localized solution structure of the FMN binding site. The electronic environment and conformation of the protein-bound flavin isoalloxazine ring were investigated by determining {sup 13}C-{sup 1}H coupling constants. The carbonyl edge of the flavin ring was found to be slightly polarized. The xylene ring was found to be nonplanar. Tyrosine 94, located adjacent to the flavin isoalloxazine ring, was shown to have a hindered aromatic ring flip rate.

  2. The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain

    PubMed Central

    Malgieri, Gaetano; Russo, Luigi; Esposito, Sabrina; Baglivo, Ilaria; Zaccaro, Laura; Pedone, Emilia M.; Di Blasio, Benedetto; Isernia, Carla; Pedone, Paolo V.; Fattorusso, Roberto

    2007-01-01

    The first putative prokaryotic Cys2His2 zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys2His2 zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys2His2 zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys2His2 zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9–66), is arranged in a βββαα topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on 15N R1, 15N R2, and heteronuclear 15N-{1H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain. PMID:17956987

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

    PubMed

    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.

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

    PubMed

    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. PMID:19107926

  5. Solution structure of the carboxyl-terminal domain of RAP74 and NMR characterization of the FCP1-binding sites of RAP74 and human TFIIB.

    PubMed

    Nguyen, Bao D; Chen, Hung-Ta; Kobor, Michael S; Greenblatt, Jack; Legault, Pascale; Omichinski, James G

    2003-02-18

    FCP1 (TFIIF-associated CTD phosphatase) is the only known phosphatase specific for the phosphorylated CTD of RNAP II. The phosphatase activity of FCP1 is strongly enhanced by the carboxyl-terminal domain of RAP74 (cterRAP74, residues 436-517), and this stimulatory effect of TFIIF can be blocked by TFIIB. It has been shown that cterRAP74 and the core domain of hTFIIB (TFIIBc, residues 112-316) directly interact with the carboxyl-terminal domain of hFCP1 (cterFCP, residues 879-961), and these interactions may be responsible for the regulatory activities of TFIIF and TFIIB on FCP1. We have determined the NMR solution structure of human cterRAP74, and we have used NMR methods to map the cterFCP-binding sites for both cterRAP74 and human TFIIB. We show that cterFCP binds to a groove of cterRAP74 between alpha-helices H2 and H3, without affecting the secondary structure of cterRAP74. We also show that cterFCP binds to a groove of TFIIBc between alpha-helices D1 and E1 in the first cyclin repeat. We find that the cterFCP-binding site of TFIIBc is very similar to the binding site for the HSV transcriptional activator protein VP16 on the first cyclin repeat of TFIIBc. The cterFCP-binding sites of both RAP74 and TFIIBc form shallow grooves on the protein surface, and they are both rich in hydrophobic and positively charged amino acid residues. These results provide new information about the recognition of acidic-rich activation domains involved in transcriptional regulation, and provide insights into how TFIIF and TFIIB regulate the FCP1 phosphatase activity in vivo. PMID:12578358

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

    PubMed

    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

  7. Variation in DNA binding constants with a change in geometry of ternary copper(II) complexes with N2O donor Schiff base and cyanate or dicyanamide

    NASA Astrophysics Data System (ADS)

    Jana, Subrata; Santra, Ramesh Chandra; Das, Saurabh; Chattopadhyay, Shouvik

    2014-09-01

    Two new copper(II) complexes, [Cu(L)(OCN)] (1) and [CuL(dca)]n (2), where HL = 2-(-(2-(diethylamino)ethylimino)methyl)naphthalen-1-ol, dca = N(CN)2-, have been synthesized and characterized by elemental analysis, IR, UV-VIS spectroscopy and single crystal X-ray diffraction studies. Complex 1 has square planar and complex 2 square pyramidal geometries in solid state around metal centre. Interactions of the complexes with calf thymus DNA (CT DNA) were studied by UV-VIS spectroscopy. Binding constant and site size of interaction were determined. Binding site size and intrinsic binding constant K revealed complex 1 interacted with calf thymus DNA better than complex 2.

  8. Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.

    PubMed

    Muñoz-García, Juan C; Chabrol, Eric; Vivès, Romain R; Thomas, Aline; de Paz, José L; Rojo, Javier; Imberty, Anne; Fieschi, Franck; Nieto, Pedro M; Angulo, Jesús

    2015-04-01

    Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns

  9. Beryllium(II) binding to ATP and ADP: potentiometric determination of the thermodynamic constants and implications for in vivo toxicity.

    PubMed

    Boukhalfa, Hakim; Lewis, James G; Crumbliss, Alvin L

    2004-04-01

    Highly toxic beryllium(II) is divalent metal ion with a high charge density, making it a potential target for binding to bio-molecules rich in O donor groups. In aqueous solution Be2+ binds to ATP and ADP to form 1:1 Be2+:ATP and Be2+:ADP complexes in relatively acidic media. At neutral pH the complex formed undergoes hydrolysis. Be2+ binding to ATP and ADP is much stronger than Ca2+ and Mg2+ binding. The high affinity of Be2+ toward ATP and ADP binding suggests a mechanism relevant to understanding the in vivo chemical toxicity of this metal.

  10. NMR investigation of the binding between human profilin I and inositol 1,4,5-triphosphate, the soluble headgroup of phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Richer, Sarah M; Stewart, Nichole K; Tomaszewski, John W; Stone, Martin J; Oakley, Martha G

    2008-12-23

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is involved in the regulation of the actin cytoskeleton through interactions with a number of actin-binding proteins. We present here NMR titration experiments that monitor the interaction between the cytoskeletal protein profilin and inositol 1,4,5-triphosphate (IP(3)), the headgroup of PI(4,5)P(2). These experiments probe the interaction directly, at equilibrium, and with profilin in its native state. We show the binding between profilin and IP(3) can readily be observed at high concentrations, even though profilin does not bind to IP(3) under physiological conditions. Moreover, the titration data using wild-type profilin and an R88L mutant support the existence of at least three headgroup binding sites on profilin, consistent with previous experimentation with intact PI(4,5)P(2). This work suggests that various soluble inositol ligands can serve as effective probes to facilitate in vitro studies of PI-binding proteins that require membrane surfaces for high-affinity binding.

  11. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  12. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor.

    PubMed

    Asencio-Hernández, Julia; Kieffer, Bruno; Delsuc, Marc-André

    2016-01-01

    There is growing evidence that bisphenol A (BPA), a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER), estrogen-related receptor (ERR) and androgen receptor (AR). BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD), which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules. PMID:27583469

  13. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor.

    PubMed

    Asencio-Hernández, Julia; Kieffer, Bruno; Delsuc, Marc-André

    2016-01-01

    There is growing evidence that bisphenol A (BPA), a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER), estrogen-related receptor (ERR) and androgen receptor (AR). BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD), which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules.

  14. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor

    PubMed Central

    Asencio-Hernández, Julia; Kieffer, Bruno

    2016-01-01

    There is growing evidence that bisphenol A (BPA), a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER), estrogen-related receptor (ERR) and androgen receptor (AR). BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD), which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules. PMID:27583469

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

    SciTech Connect

    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.

  16. NMR Studies of Heat-Induced Transitions in Structure and Cation Binding Environments of a Strontium-Saturated Swelling Mica

    SciTech Connect

    Bowers, Geoffrey M.; Davis, Michael C.; Ravella, Ramesh; Komarneni, S.; Mueller, Karl T.

    2007-12-03

    In this work we combined Al, Si, F, and Na magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to characterize the structure and interlayer cation environments in a strontium-saturated member of the swelling mica family before and after a heat induced collapse of the interlayer space.

  17. H-H, C-H, and C-C NMR spin-spin coupling constants calculated by the FP-INDO method for aromatic hydrocarbons

    NASA Technical Reports Server (NTRS)

    Long, S. A. T.; Memory, J. D.

    1978-01-01

    The FP-INDO (finite perturbation-intermediate neglect of differential overlap) method is used to calculate the H-H, C-H, and C-C coupling constants in hertz for molecules of six different benzenoid hydrocarbons: benzene, naphthalene, biphenyl, anthracene, phenanthrene, and pyrene. The calculations are based on both the actual and the average molecular geometries. It is found that only the actual molecular geometries can always yield the correct relative order of values for the H-H coupling constants. For the calculated C-C coupling constants, as for the calculated C-H coupling constants, the signs are positive (negative) for an odd (even) number of bonds connecting the two nuclei. Agreements between the calculated and experimental values of the coupling constants for all six molecules are comparable to those reported previously for other molecules.

  18. 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

    SciTech Connect

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

    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.

  19. Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: The inhibitory effect of linoleic acid on the DNA-binding step

    SciTech Connect

    Jung, Kyung Chae; Rhee, Ho Sung; Park, Chi Hoon; Yang, Chul-Hak . E-mail: chulyang@plaza.snu.ac.kr

    2005-08-19

    c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90({+-}0.54) x 10{sup -7} M for Max85/Max85 homodimer, 6.85({+-}0.25) x 10{sup -3} M for Myc87/Myc87 homodimer, and 2.55({+-}0.29) x 10{sup -8} M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33({+-}0.21) x 10{sup -9} M for Max85/Max85/DNA, 2.27({+-}0.08) x 10{sup -12} M for Myc87/Myc87/DNA, and 4.43({+-}0.37) x 10{sup -10} M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.

  20. An introduction to biological NMR spectroscopy.

    PubMed

    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).

  1. An Introduction to Biological NMR Spectroscopy*

    PubMed Central

    Marion, Dominique

    2013-01-01

    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). PMID:23831612

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

    PubMed

    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).

  3. Combined X-ray, NMR, and Kinetic Analyses Reveal Uncommon Binding Characteristics of the Hepatitis C Virus NS3-NS4A Protease Inhibitor BI 201335*

    PubMed Central

    Lemke, Christopher T.; Goudreau, Nathalie; Zhao, Songping; Hucke, Oliver; Thibeault, Diane; Llinàs-Brunet, Montse; White, Peter W.

    2011-01-01

    Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with direct-acting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a single-step binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine–oxygen halogen bond, and profound pKa changes within the catalytic triad associated with binding of the bromo-quinoline moiety. PMID:21270126

  4. Solution-state NMR Investigation of DNA Binding Interactions in Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg): A Dynamic Description of the DNA/Protein Interface

    SciTech Connect

    Buchko, Garry W.; McAteer, Kathleen; Wallace, Susan S.; Kennedy, Michael A.

    2005-03-02

    Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free and bound to a double-stranded DNA oligomer (13-PD) containing propanediol, a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 252 (83%) free-precession HSQC cross peaks were observed and 180 of these were assignable, indicating that ~30% of the residues undergo intermediate timescale motion that makes them intractable in backbone assignment experiments. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate time-scale motion observed for free Fpg. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that was quenched upon binding 13-PD. Collectively, these observations reveal that, in solution, Fpg contains highly flexible regions. The dynamic nature of Fpg, especially at the DNA binding surface, may be key to its processive search mechanism.

  5. NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

    PubMed Central

    Bycroft, M; Grünert, S; Murzin, A G; Proctor, M; St Johnston, D

    1995-01-01

    The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way. Images PMID:7628456

  6. Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.

    PubMed

    Olson, Linda J; Jensen, Davin R; Volkman, Brian F; Kim, Jung-Ja P; Peterson, Francis C; Gundry, Rebekah L; Dahms, Nancy M

    2015-07-01

    The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Å resolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches. PMID:25863146

  7. Diminazene or berenil, a classic duplex minor groove binder, binds to G-quadruplexes with low nanomolar dissociation constants and the amidine groups are also critical for G-quadruplex binding.

    PubMed

    Zhou, Jie; Le, Vu; Kalia, Dimpy; Nakayama, Shizuka; Mikek, Clinton; Lewis, Edwin A; Sintim, Herman O

    2014-10-01

    G-quadruplexes have shown great promise as chemotherapeutic targets, probably by inhibiting telomere elongation or downregulating oncogene expression. There have been many G-quadruplex ligands developed over the years but only a few have drug-like properties. Consequently only a few G-quadruplex ligands have entered clinical trials as cancer chemotherapeutic agents. The DNA minor groove ligand, berenil (diminazene aceturate or DMZ), is used to treat animal trypanosomiasis and hence its toxicological profile is already known, making it an ideal platform to engineer into new therapeutics. Herein, using a plethora of biophysical methods including UV, NMR, MS and ITC, we show that DMZ binds to several G-quadruplexes with a Kd of ∼1 nM. This is one of the strongest G-quadruplex binding affinities reported to date and is 10(3) tighter than the berenil affinity for an AT-rich duplex DNA. Structure-activity-relationship studies demonstrate that the two amidine groups on DMZ are important for binding to both G-quadruplex and duplex DNA. This work reveals that DMZ or berenil is not as selective for AT-rich duplexes as originally thought and that some of its biological effects could be manifested through G-quadruplex binding. The DMZ scaffold represents a good starting point to develop new G-quadruplex ligands for cancer cell targeting. PMID:25096593

  8. NMR studies of metalloproteins.

    PubMed

    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.

  9. Determination of the ratio of cationic flexible nanoparticles binding constant of counterions X- and Br- (KX/KBr) using the semi-empirical spectrophotometric (SESp) technique

    NASA Astrophysics Data System (ADS)

    Khalid, Khalisanni; Khan, M. Niyaz; Sharifuddin, M. Z.; Kim, R. P. T.; Azri, M. N. Muhammad

    2016-07-01

    The determination of the ion exchange constant (KXBr) for ion-exchange process occurring between counterions X- and Br- (X= C6H5CO2- ) at the cationic flexible nanoparticles surface using the semi-emperical spectrophotometric (SESp) technique is described in this paper. The method uses an anionic spectrophotometric probe molecule, N-(2-methoxyphnyl) phthalamate ion (1-), which measures the effects of varying concentrations of nanoparticles (CTABr/NaX/H2O) on absorbance, (Aob) at 310 nm of samples containing constant concentration of (1-), NaOH, CTABr and NaX. The observed data, Aob vs [NaX] fit satisfactorily to an empirical equation which gives the values of two empirical constants. These empirical constants lead to the determination of KXBr (=KX/KBr with KX and KBr representing cationic flexible nanoparticles binding constants of counterions X- Br- respectively). This method gives values of KXBr for moderately hydrophobic X-. The value of KXBr, obtained by using this method, is comparable with the corresponding values of KXBr , obtained by the use of semi-emperical kinetic (SEK) method.

  10. Molecular environment of stable iodine and radioiodine (129I) in natural organic matter: Evidence inferred from NMR and binding experiments at environmentally relevant concentrations

    NASA Astrophysics Data System (ADS)

    Xu, Chen; Zhong, Junyan; Hatcher, Patrick G.; Zhang, Saijin; Li, Hsiu-Ping; Ho, Yi-Fang; Schwehr, Kathleen A.; Kaplan, Daniel I.; Roberts, Kimberly A.; Brinkmeyer, Robin; Yeager, Chris M.; Santschi, Peter H.

    2012-11-01

    129I is a major by-product of nuclear fission and had become one of the major radiation risk drivers at Department of Energy (DOE) sites. 129I is present at elevated levels in the surface soils of the Savannah River Site (SRS) F-Area and was found to be bound predominantly to soil organic matter (SOM). Naturally bound 127I and 129I to sequentially extracted humic acids (HAs), fulvic acids (FAs) and a water extractable colloid (WEC) were measured in a 129I-contaminated wetland surface soil located on the SRS. WEC is a predominantly colloidal organic fraction obtained from soil re-suspension experiments to mimic the fraction that may be released during groundwater exfiltration, storm water or surface runoff events. For the first time, NMR techniques were applied to infer the molecular environment of naturally occurring stable iodine and radioiodine binding to SOM. Iodine uptake partitioning coefficients (Kd) by these SOM samples at ambient iodine concentrations were also measured and related to quantitative structural analyses by 13C DPMAS NMR and solution state 1H NMR on the eight humic acid fractions. By assessing the molecular environment of iodine, it was found that it was closely associated with the aromatic regions containing esterified products of phenolic and formic acids or other aliphatic carboxylic acids, amide functionalities, quinone-like structures activated by electron-donating groups (e.g., NH2), or a hemicellulose-lignin-like complex with phenyl-glycosidic linkages. However, FAs and WEC contained much greater concentrations of 127I or 129I than HAs. The contrasting radioiodine contents among the three different types of SOM (HAs, FAs and WEC) suggest that the iodine binding environment cannot be explained solely by the difference in the amount of their reactive binding sites. Instead, indirect evidence indicates that the macro-molecular conformation, such as the hydrophobic aliphatic periphery hindering the active aromatic cores and the hydrophilic

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

    ERIC Educational Resources Information Center

    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;…

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

    PubMed

    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. PMID:27112228

  13. NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

    PubMed

    D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

    2009-01-01

    The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed. PMID:18977333

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

    PubMed

    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. NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

    PubMed

    D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

    2009-01-01

    The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

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

    PubMed

    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. PMID:27422118

  17. Fluorine detected 2D NMR experiments for the practical determination of size and sign of homonuclear F-F and heteronuclear C-F multiple bond J-coupling constants in multiple fluorinated compounds

    NASA Astrophysics Data System (ADS)

    Aspers, Ruud L. E. G.; Ampt, Kirsten A. M.; Dvortsak, Peter; Jaeger, Martin; Wijmenga, Sybren S.

    2013-06-01

    The use of fluorine in molecules obtained from chemical synthesis has become increasingly important within the pharmaceutical and agricultural industry. NMR characterization of these compounds is of great value with respect to their structure elucidation, their screening in metabolomics investigations and binding studies. The favorable NMR properties of the fluorine nucleus make NMR with fluorine detection of great value in this respect. A suite of NMR 2D F-F- and F-C-correlation experiments with fluorine detection was applied to the assignment of resonances, nJCF- and nJFF-couplings as well as the determination of their size and sign. The utilization of this experiment suite was exemplarily demonstrated for a highly fluorinated vinyl alkyl ether. Especially F-C HSQC and J-scaled F-C HMBC experiments allowed determining the size of the J-couplings of this compound. The relative sign of its homo- and heteronuclear couplings was achieved by different combinations of 2D NMR experiments, including non-selective and F2-selective F-C XLOC, F2-selective F-C HMQC, and F-F COSY. The F2-one/two-site selective F-C XLOC versions were found highly useful, as they led to simplifications of the common E.COSY patterns and resulted in a higher confidence level of the assignment by using selective excitation. The combination of F2-one/two-site selective F-C XLOC experiments with a F2-one-site selective F-C HMQC experiment provided the signs of all nJCF- and nJFF-couplings in the vinyl moiety of the test compound. Other combinations of experiments were found useful as well for special purposes when focusing for example on homonuclear couplings a combination of F-F COSY-10 with a F2-one-site selective F-C HMQC could be used. The E.COSY patterns in the spectra demonstrated were analyzed by use of the spin-selective displacement vectors, and in case of the XLOC also by use of the DQ- and ZQ-displacement vectors. The variety of experiments presented shall contribute to facilitate the

  18. Solution conformation of a peptide fragment representing a proposed RNA-binding site of a viral coat protein studied by two-dimensional NMR

    SciTech Connect

    van der Graaf, M.; van Mierlo, C.P.M.; Hemminga, M.A. )

    1991-06-11

    The first 25 amino acids of the coat protein of cowpea chlorotic mottle virus are essential for binding the encapsidated RNA. Although an {alpha}-helical conformation has been predicted for this highly positively charged N-terminal region. No experimental evidence for this conformation has been presented so far. In this study, two-dimensional proton NMR experiments were performed on a chemically synthesized pentacosapeptide containing the first 25 amino acids of this coat protein. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly converting into more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium toward more helical structures. At 10{degrees}C, a perceptible fraction of the conformational ensemble consists of structures with an {alpha}-helical conformation between residues 9 and 17, likely starting with a turnlike structure around Thr9 and Arg10. Both the conformation and the position of this helical region agree well with the secondary structure predictions mentioned above.

  19. Complex formation of fenchone with α-cyclodextrin: NMR titrations.

    PubMed

    Nowakowski, Michał; Ejchart, Andrzej

    2014-01-01

    (13)C NMR titration studies of inclusion complexes of bicyclic terpenoid, fenchone enantiomers with α-cyclodextrin revealed their 1:2 guest-host stoichiometry. Sequential binding constants were determined indicating a strong binding cooperativity of two α-cyclodextrin to fenchone. The overall association constants were used to calculate the Gibbs free energies of diastereomeric complex formation, which might be used as a measure of chiral recognition of fenchone by α-cyclodextrin. These results were compared with corresponding data derived for camphor, which is an isomeric bicyclic terpenoid.

  20. Toll-like receptors (TLRs) and mannan-binding lectin (MBL): on constant alert in a hostile environment.

    PubMed

    Bergman, Ingrid-Maria

    2011-05-01

    In the beginning were neither B cells nor T cells nor antibodies, but innate immune defense alone. The primary functional theme of innate immunity is the distinction between self and non-self, which is maintained by a vast number of cellular and subcellular components. In this context, the immense importance of the Toll-like receptors (TLRs) is well established. Positive (Darwinian) selection seems to be acting on the ligand-binding domains of these molecules, suggesting a selection pattern similar to that previously observed in the MHC proteins. In sharp contrast to TLRs, the biological significance of mannan-binding lectin (MBL) is controversial, and, concerning humans, it has been suggested that low concentration of MBL in serum represents a selective advantage. In this mini-review, based on a doctoral thesis, evolutionary aspects of TLRs and MBL are discussed. PMID:21323627

  1. NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1.

    PubMed

    Omichinski, J G; Clore, G M; Schaad, O; Felsenfeld, G; Trainor, C; Appella, E; Stahl, S J; Gronenborn, A M

    1993-07-23

    The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone. PMID:8332909

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

    PubMed

    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.

  3. Complexation-flocculation combined with microwave-assisted headspace solid-phase microextraction in determining the binding constants of hydrophobic organic pollutants to dissolved humic substances.

    PubMed

    Hsieh, Ping-Chieh; Lee, Chon-Lin; Jen, Jen-Fon; Chang, Kuei-Chen

    2015-02-21

    The binding constants, KDOC, of selected polycyclic aromatic hydrocarbons (PAHs)-phenanthrene, anthracene, fluoranthene, and pyrene-to dissolved humic substances (DHS) were determined by complexation-flocculation combined with microwave-assisted headspace solid-phase microextraction (CF-MA-HS-SPME). The results obtained are comparable with KDOC data reported in the literature. No disruption of the PAH to DHS binding equilibrium was observed during the complexation-flocculation process. The present study, which is the first to determine KDOC by CF-MA-HS-SPME, provides an alternative approach to determine the KDOC of PAHs. CF-MA-HS-SPME provides some advantages over other methods, such as no limitation of fluorescent compounds, greater determination speed, and the capability of measuring various compounds simultaneously.

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

    PubMed

    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.

  5. The Potential Utility of Predicted One Bond Carbon-Proton Coupling Constants in the Structure Elucidation of Small Organic Molecules by NMR Spectroscopy

    PubMed Central

    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. PMID:25365289

  6. Inclusion complexes of PBN-type nitrone spin traps and their superoxide spin adducts with cyclodextrin derivatives: parallel determination of the association constants by NMR titrations and 2D-EPR simulations.

    PubMed

    Bardelang, David; Rockenbauer, Antal; Karoui, Hakim; Finet, Jean-Pierre; Tordo, Paul

    2005-05-26

    (1)H NMR and electron paramagnetic resonance (EPR) titrations were used to determine the association constants of the complexes of alpha-phenyl-N-tert-butylnitrone (PBN) analogues and their superoxide spin adducts, respectively, with methylated beta-cyclodextrins. A 1:1 stoichiometry for the nitrones with randomly methylated beta-cyclodextrin and 2,6-di-O-methyl-beta-cyclodextrin and 1:1 and 1:2 stoichiometries for the corresponding cyclodextrin-nitroxide complexes were observed. After the superoxide radical spin trapping reaction, EPR titrations afforded the association constants of the corresponding cyclodextrin-nitroxide complexes. Two-dimensional EPR simulations indicated a bimodal inclusion of the nitroxide free radical spin adducts into the cyclodextrins. For all the nitrone-cyclodextrin and nitroxide-cyclodextrin complexes, the association constants were always higher for the nitroxide complexes than for the nitrone complexes. A cooperative system concerning the complexation of the nitroxide spin adduct with a cyclodextrin was evidenced by EPR titrations. The efficiency of the cyclodextrin inclusion technique to trap superoxide and to resist bioreduction by sodium l-ascorbate was also investigated.

  7. A Large Intrinsically Disordered Region in SKIP and Its Disorder-Order Transition Induced by PPIL1 Binding Revealed by NMR*

    PubMed Central

    Wang, Xingsheng; Zhang, Shaojie; Zhang, Jiahai; Huang, Xiaojuan; Xu, Chao; Wang, Weiwei; Liu, Zhijun; Wu, Jihui; Shi, Yunyu

    2010-01-01

    Intrinsically disordered proteins or protein regions play an important role in fundamental biological processes. During spliceosome activation, a large structural rearrangement occurs. The Prp19 complex and related factors are involved in the catalytic activation of the spliceosome. Recent mass spectrometric analyses have shown that Ski interaction protein (SKIP) and peptidylprolyl isomerase-like protein 1 (PPIL1) are Prp19-related factors that constitute the spliceosome B, B*, and C complexes. Here, we report that a highly flexible region of SKIP (SKIPN, residues 59–129) is intrinsically disordered. Upon binding to PPIL1, SKIPN undergoes a disorder-order transition. A highly conserved fragment of SKIP (residues 59–79) called the PPIL1-binding fragment (PBF) was sufficient to bind PPIL1. The structure of PBF·PPIL1 complex, solved by NMR, shows that PBF exhibits an ordered structure and interacts with PPIL1 through electrostatic and hydrophobic interactions. Three subfragments in the PBF (residues 59–67, 68–73, and 74–79) show hook-like backbone structure, and interactions between these subfragments are necessary for PBF·PPIL1 complex formation. PPIL1 is a cyclophilin family protein. It is recruited by SKIP into the spliceosome by a region other than the peptidylprolyl isomerase active site. This enables the active site of PPIL1 to remain open in the complex and still function as a peptidylprolyl cis/trans-isomerase or molecular chaperon to facilitate the folding of other proteins in the spliceosomes. The large disordered region in SKIP provides an interaction platform. Its disorder-order transition, induced by PPIL1 binding, may adapt the requirement for a large structural rearrangement occurred in the activation of spliceosome. PMID:20007319

  8. 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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    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 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.

  9. 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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA

    PubMed Central

    Zhuang, Jianqin; Yang, De-Ping; Tian, Xiaoyu; Nikas, Spyros P.; Sharma, Rishi; Guo, Jason Jianxin; Makriyannis, Alexandros

    2013-01-01

    Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using 19F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain. PMID:24533425

  12. Some critical aspects in the determination of binding constants by electrospray ionisation mass spectrometry at the example of arsenic bindings to sulphur-containing biomolecules.

    PubMed

    Schmidt, Anne-Christine; Steier, Sandra

    2010-08-01

    The influences of reactant concentrations, solvent type, acid strength, pH conditions and ionic strength on the determination of apparent gas-phase equilibrium constants K using electrospray ionisation mass spectrometry (ESI-MS) were elucidated. As example serves the interaction of the tripeptide glutathione (GSH) with phenylarsine oxide (PAO). It was shown that rising initial concentrations of both reactants were not adequately compensated by increasing signal intensities of the reaction products in the mass spectra. The equilibrium constant for the formation of the phenylarsenic-substituted peptide species decreased from 1.42 x 10(5) +/- 1.81 x 10(4) l micromol(-1) to 1.54 x 10(4) +/- 1.5 x 10(3) l micromol(-1) with rising initial GSH concentrations from 1 to 10 microM at fixed PAO molarity of 50 microM. K values resulting from a series with a fixed GSH molarity of 5 microM and a PAO molarity varied from 10 to 100 microM remained in a narrower range between 4.59 x 10(4) +/- 2.15 x 10(4) l micromol(-1) and 1.07 x 10(4) +/- 4.0 x 10(3) l micromol(-1). In contrast, consumption numbers calculated from the ion intensity ratios of reaction products to the unreacted peptide were not influenced by the initial reactant concentrations. In a water-acetonitrile-acetic acid mixture (48:50:2, v:v), the consumption of 5 micro M GSH increased from 8.3 +/- 1.4% to 39.6 +/- 1.6% with increased molar excess of PAO from 2 to 20, respectively. The GSH consumption was considerably enhanced in a changed solvent system consisting of 25% acetonitrile and 75% 10 mM ammonium formate, pH 5.0 (v:v) up to 80% of the original peptide amount at an only threefold molar arsenic excess.

  13. Intrinsic propensities of amino acid residues in GxG peptides inferred from amide I' band profiles and NMR scalar coupling constants.

    PubMed

    Hagarman, Andrew; Measey, Thomas J; Mathieu, Daniel; Schwalbe, Harald; Schweitzer-Stenner, Reinhard

    2010-01-20

    A reliable intrinsic propensity scale of amino acid residues is indispensable for an assessment of how local conformational distributions in the unfolded state can affect the folding of peptides and proteins. Short host-guest peptides, such as GxG tripeptides, are suitable tools for probing such propensities. To explore the conformational distributions sampled by the central amino acid residue in these motifs, we combined vibrational (IR, Raman, and VCD) with NMR spectroscopy. The data were analyzed in terms of a superposition of two-dimensional Gaussian distribution functions in the Ramachandran space pertaining to subensembles of polyproline II, beta-strand, right- and left-handed helical, and gamma-turn-like conformations. The intrinsic propensities of eight amino acid residues (x = A, V, F, L, S, E, K, and M) in GxG peptides were determined as mole fractions of these subensembles. Our results show that alanine adopts primarily (approximately 80%) a PPII-like conformation, while valine and phenylalanine were found to sample PPII and beta-strand-like conformations equally. The centers of the respective beta-strand distributions generally do not coincide with canonical values of dihedral angles of residues in parallel or antiparallel beta-strands. In fact, the distributions for most residues found in the beta-region significantly overlap the PPII-region. A comparison with earlier reported results for trivaline reveals that the terminal valines increase the beta-strand propensity of the central valine residue even further. Of the remaining investigated amino acids, methionine preferred PPII the most (0.64), and E, S, L, and K exhibit moderate (0.56-0.45) PPII propensities. Residues V, F, S, E, and L sample, to a significant extent, a region between the canonical PPII and (antiparallel) beta-strand conformations. This region coincides with the sampling reported for L and V using theoretical predictions (Tran et al. Biochemistry 2005, 44, 11369). The distributions of

  14. Quantitative chemical exchange saturation transfer with hyperpolarized nuclei (qHyper-CEST): Sensing xenon-host exchange dynamics and binding affinities by NMR

    SciTech Connect

    Kunth, M. Witte, C.; Schröder, L.

    2014-11-21

    The reversible binding of xenon to host molecules has found numerous applications in nuclear magnetic resonance studies. Quantitative characterization of the Xe exchange dynamics is important to understand and optimize the physico-chemical behavior of such Xe hosts, but is often challenging to achieve at low host concentrations. We have investigated a sensitive quantification technique based on chemical exchange saturation transfer with hyperpolarized nuclei, qHyper-CEST. Using simulated signals we demonstrated that qHyper-CEST yielded accurate and precise results and was robust in the presence of large amounts of noise (10%). This is of particular importance for samples with completely unknown exchange rates. Using these findings we experimentally determined the following exchange parameters for the Xe host cryptophane-A monoacid in dimethyl sulfoxide in one type of experiment: the ratio of bound and free Xe, the Xe exchange rate, the resonance frequencies of free and bound Xe, the Xe host occupancy, and the Xe binding constant. Taken together, qHyper-CEST facilitates sensitive quantification of the Xe exchange dynamics and binding to hydrophobic cavities and has the potential to analyze many different host systems or binding sites. This makes qHyper-CEST an indispensable tool for the efficient design of highly specific biosensors.

  15. Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR

    PubMed Central

    Monleón, Daniel; Esteve, Vicent; Kovacs, Helena; Calvete, Juan J.; Celda, Bernardo

    2004-01-01

    Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30°. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin–αvβ3 docking model suggests that echistatin's C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function correlations. PMID:15535803

  16. Configuration interaction calculations of positron binding to molecular oxides and hydrides and its effect on spectroscopic constants

    NASA Astrophysics Data System (ADS)

    Buenker, Robert J.; Liebermann, Heinz-Peter

    2008-02-01

    Ab initio multireference single- and double-excitation configuration interaction (MRD-CI) calculations have been carried out for magnesium oxide (MgO) and lithium oxide (LiO) and their positronic complexes. These results are compared with previous theoretical data obtained earlier for beryllium oxide (BeO) and the series of alkali hydrides with and without an additional positron. Potential curves have been constructed for each of the systems, MgO, e +MgO, LiO and e +LiO. Positron affinities (PAs) of 0.472 eV and 0.304 eV, respectively, have been computed for the ground states of MgO and LiO. Because of the relatively low ionization potential of the Li atom, it is found that the dissociation limit in the latter case is Li + + PsO (Oe +e -), whereas it is Mg + e +O in the former case, in close analogy to what has been found for e +BeO. Significant changes in bond lengths, vibrational frequencies and dissociation energies are indicated as a result of binding a positron to each of these oxides as well as to the alkali hydrides studied earlier. The general trend observed is toward increased bond length and decreased frequency as a result of attaching a positron to these systems in their various low-lying electronic states.

  17. From correlation-consistent to polarization-consistent basis sets estimation of NMR spin spin coupling constant in the B3LYP Kohn Sham basis set limit

    NASA Astrophysics Data System (ADS)

    Kupka, Teobald

    2008-08-01

    Based on B3LYP spin-spin coupling constants (SSCC) of several molecules calculated with cc-pV xZ, cc-pCV xZ, cc-pCV xZ-sd and cc-pCV xZ-sd+ t basis sets, a reasonably fit, using the two-parameter formula, to the Kohn-Sham complete basis set limit (CBS) is shown. Improvement in the CBS values going from cc-pV xZ to the most elaborated cc-pCV xZ-sd+ t basis set family is observed: standard deviation for all data drops from 33.7 to 23.1, and from 6.0 to 4.8 Hz after excluding problematic 1J(F,H) and 1J(F,C). Calculation of water's 1J(OH) using B3LYP/cc-pCV xZ and B3LYP/pcJ- n significantly improved the FC term convergence.

  18. Characterizing carbohydrate-protein interactions by NMR

    PubMed Central

    Bewley, Carole A.; Shahzad-ul-Hussan, Syed

    2013-01-01

    Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792

  19. Norfloxacin Zn(II)-based complexes: acid base ionization constant determination, DNA and albumin binding properties and the biological effect against Trypanosoma cruzi.

    PubMed

    Gouvea, Ligiane R; Martins, Darliane A; Batista, Denise da Gama Jean; Soeiro, Maria de Nazaré C; Louro, Sonia R W; Barbeira, Paulo J S; Teixeira, Letícia R

    2013-10-01

    Zn(II) complexes with norfloxacin (NOR) in the absence or in the presence of 1,10-phenanthroline (phen) were obtained and characterized. In both complexes, the ligand NOR was coordinated through a keto and a carboxyl oxygen. Tetrahedral and octahedral geometries were proposed for [ZnCl2(NOR)]·H2O (1) and [ZnCl2(NOR)(phen)]·2H2O (2), respectively. Since the biological activity of the chemicals depends on the pH value, pH titrations of the Zn(II) complexes were performed. UV spectroscopic studies of the interaction of the complexes with calf-thymus DNA (CT DNA) have suggested that they can bind to CT DNA with moderate affinity in an intercalative mode. The interactions between the Zn(II) complexes and bovine serum albumin (BSA) were investigated by steady-state and time-resolved fluorescence spectroscopy at pH 7.4. The experimental data showed static quenching of BSA fluorescence, indicating that both complexes bind to BSA. A modified Stern-Volmer plot for the quenching by complex 2 demonstrated preferential binding near one of the two tryptophan residues of BSA. The binding constants obtained (K b ) showed that BSA had a two orders of magnitude higher affinity for complex 2 than for 1. The results also showed that the affinity of both complexes for BSA was much higher than for DNA. This preferential interaction with protein sites could be important to their biological mechanisms of action. The analysis in vitro of the Zn(II) complexes and corresponding ligand were assayed against Trypanosoma cruzi, the causative agent of Chagas disease and the data showed that complex 2 was the most active against bloodstream trypomastigotes.

  20. 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.

    PubMed

    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.

  1. 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.

    PubMed

    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.

  2. High resolution 2D-NMR studies indicating complete assignments and conformational characteristics of the NF-kappa B binding enhancer element of HIV-LTR.

    PubMed

    Singh, M P; Fregeau, N L; Pon, R T; Lown, J W

    1995-10-01

    The asymmetrical DNA duplex [5'd(AAGGGACTTTCC)].[5'-d(GGAAAGTCCCTT)] has been studied by one- and two-dimensional NMR techniques. The sequence is comprised of the actual 10 base-pair long binding site for the transcription factor NF-kappa B in the enhancer sequence of the long term repeat (LTR) region of HIV and SIV types of retroviruses associated with the AIDS syndrome. Two additional A.T base-pairs are also included on one end for an added interest in the 12-bp duplex sequence with a pseudo dyad-symmetric disposition of the oligopurine and oligopyrimidine segments, as it appears in the HIV-1 genome. Phase-sensitive two-dimensional spectra (NOESY, ROESY, COSY and TOCSY) were obtained at three different temperatures (5, 15 and 25 degrees C) for a complete assignment of the non-exchangeable protons by tracing through sequence specific intra- and internucleotide connectivities. 2D-NOESY spectra were also acquired in aqueous (90% H2O-D2O) solutions, with two different methods of water signal suppression, to assign the exchangeable protons from specific NOE correlations. Adenine H2 protons were assigned by the use of NOE correlations and from T1 relaxation time measurements. The general spectral features and semi-quantitative interproton distance estimates indicate a B-DNA type conformation. However, some distinctly unusual features associated with the nucleotides at and immediately adjacent to both the 5'-and 3'-ends of AAA/TTT and GGG/CCC segments were noted. The complete assignments, and the observed characteristics, will be of significant value in studying the complexes of this transcriptionally active DNA domain with the protein and other rationally designed DNA binding agents.

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

    PubMed

    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.

  4. Determination of the lithium binding site in inositol monophosphatase, the putative target for lithium therapy, by magic-angle-spinning solid-state NMR.

    PubMed

    Haimovich, Anat; Eliav, Uzi; Goldbourt, Amir

    2012-03-28

    Inositol monophosphatase (IMPase) catalyzes the hydrolysis of inositol monophosphate to inorganic phosphate and inositol. For this catalytic process to occur, Mg(2+) cations must exist in the active site. According to the inositol depletion hypothesis, IMPase activity is assumed to be higher than normal in patients suffering from bipolar disorder. Treatment with Li(+), an inhibitor of IMPase, reduces its activity, but the mechanism by which lithium exerts its therapeutic effects is still at a stage of conjecture. The Escherichia coli SuhB gene product possesses IMPase activity, which is also strongly inhibited by Li(+). It has significant sequence similarity to human IMPase and has most of its key active-site residues. Here we show that by using (7)Li magic-angle-spinning solid-state NMR spectroscopy, including {(13)C}(7)Li dipolar recoupling experiments, the bound form of lithium in the active site of wild-type E. coli SuhB can be unambiguously detected, and on the basis of our data and other biochemical data, lithium binds to site II, coupled to aspartate residues 84, 87, and 212.

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

    PubMed

    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.

  6. Label-free determination of protein-ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry.

    PubMed

    Jecklin, Matthias C; Schauer, Stefan; Dumelin, Christoph E; Zenobi, Renato

    2009-01-01

    We performed a systematic comparison of three label-free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI-MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K(D)). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946-1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K(D) value determined for ethoxzolamide by nESI-MS was 5 +/- 1 nM and the K(D) value for sulfanilamide was 145.7 +/- 10.0 microM. The agreement of the determined K(D) values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4-carboxybenzenesulfonamide (variation by approximately one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K(D) values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. PMID:19373858

  7. Study of stereospecificity of 1H, 13C, 15N and 77Se shielding constants in the configurational isomers of the selenophene-2-carbaldehyde azine by NMR spectroscopy and MP2-GIAO calculations.

    PubMed

    Afonin, Andrei V; Pavlov, Dmitry V; Albanov, Alexander I; Levanova, Ekaterina P; Levkovskaya, Galina G

    2011-11-01

    In the (1)H and (13)C NMR spectra of selenophene-2-carbaldehyde azine, the (1)H-5, (13)C-3 and (13)C-5 signals of the selenophene ring are shifted to higher frequencies, whereas those of the (1)H-1, (13)C-1, (13)C-2 and (13)C-4 are shifted to lower frequencies on going from the EE to ZZ isomer or from the E moiety to the Z moiety of EZ isomer. The (15)N chemical shift is significantly larger in the EE isomer relative to the ZZ isomer and in the E moiety relative to the Z moiety of EZ isomer. A very pronounced difference (60-65 mg/g) between the (77)Se resonance positions is revealed in the studied azine isomers, the (77)Se peak being shifted to higher frequencies in the ZZ isomer and in the Z moiety of EZ isomer. The trends in the changes of the measured chemical shifts are reasonably reproduced by the GIAO calculations at the MP2 level of the (1)H, (13)C, (15)N and (77)Se shielding constants in the energy-favorable conformation with the syn orientation of both selenophene rings relative to the C = N groups. The NBO analysis suggests that such an arrangement of the selenophene rings may take place because of a higher energy of some intramolecular interactions. PMID:22002712

  8. Determination of the tautomeric equilibria of pyridoyl benzoyl β-diketones in the liquid and solid state through the use of deuterium isotope effects on (1)H and (13)C NMR chemical shifts and spin coupling constants.

    PubMed

    Hansen, Poul Erik; Borisov, Eugeny V; Lindon, John C

    2015-02-01

    The tautomeric equilibria for 2-pyridoyl-, 3-pyridoyl-, and 4-pyridoyl-benzoyl methane have been investigated using deuterium isotope effects on (1)H and (13)C chemical shifts both in the liquid and the solid state. Equilibria are established both in the liquid and the solid state. In addition, in the solution state the 2-bond and 3-bond J((1)H-(13)C) coupling constants have been used to confirm the equilibrium positions. The isotope effects due to deuteriation at the OH position are shown to be superior to chemical shift in determination of equilibrium positions of these almost symmetrical -pyridoyl-benzoyl methanes. The assignments of the NMR spectra are supported by calculations of the chemical shifts at the DFT level. The equilibrium positions are shown to be different in the liquid and the solid state. In the liquid state the 4-pyridoyl derivative is at the B-form (C-1 is OH), whereas the 2-and 3-pyridoyl derivatives are in the A-form. In the solid state all three compounds are on the B-form. The 4-pyridoyl derivative shows unusual deuterium isotope effects in the solid, which are ascribed to a change of the crystal structure of the deuteriated compound. PMID:24070650

  9. 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.

    PubMed

    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.

  10. Revised stability constant, spectroscopic properties and binding mode of Zn(II) to FluoZin-3, the most common zinc probe in life sciences.

    PubMed

    Marszałek, I; Krężel, A; Goch, W; Zhukov, I; Paczkowska, I; Bal, W

    2016-08-01

    2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant ((C)Kd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the (C)Kd for Zn(II) complexation by FluoZin-3 at pH7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV-Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight pKa values from fluorescence spectra and from various regions of UV-Vis spectra. The validated values of (C)Kd (9.1±0.4nM; -log (C)Kd=8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically. PMID:27216451

  11. Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

    PubMed Central

    Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

    1999-01-01

    A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Sequence-specific sup 1 H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX

    SciTech Connect

    Huang, L.H.; Cheng, H.; Sweeney, W.V. ); Pardi, A. ); Tam, J.P. )

    1991-07-30

    Factor IX is a blood clotting protein that contains three regions, including a {gamma}-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF0like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel {beta}-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp{sup 28}, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the {beta}-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.

  14. Breaking pseudo-symmetry in multiantennary complex N-glycans using lanthanide-binding tags and NMR pseudo-contact shifts.

    PubMed

    Canales, Angeles; Mallagaray, Alvaro; Pérez-Castells, Javier; Boos, Irene; Unverzagt, Carlo; André, Sadine; Gabius, Hans-Joachim; Cañada, Francisco Javier; Jiménez-Barbero, Jesús

    2013-12-16

    Controlling NMR shifts by lanthanides tagged to a "symmetrical" N-glycan reveals individual resonances for the residues of the otherwise identical A and B arms. This method provides a global perspective of conformational features and interactions in solution.

  15. pH and ionic strength effects on the binding constant between a nitrogen-containing polycyclic aromatic compound and humic acid.

    PubMed

    Chang, Kuei-Chen; Lee, Chon-Lin; Hsieh, Ping-Chieh; Brimblecombe, Peter; Kao, Shu-Min

    2015-09-01

    Polycyclic aromatic compounds (PACs) are widespread environmental pollutants with a high potential to act as human carcinogens and mutagens. The behavior of PACs is significantly affected by their interactions with dissolved organic matter (DOM), such as their transport, solubility, bioavailability, and bioaccumulation in the aquatic environment. Being a basic PAC, benzo(h)quinoline (BQ) is the dominant species, as the solution's pH value is higher than BQ's pK a (pK a of BQ = 4.2). In contrast, benzo(h)quinolinium (BQH(+)) is the major species, as the solution's pH value is lower than its pK a. The binding constant (K DOC), measured by fluorescence quenching, between BQ/BQH(+) and Leonardite humic acid (LHA) would decrease 70 to 95 % and 20 to 90 % when increasing the ionic strength in acidic and neutral to basic conditions, respectively. The results can be attributed to the added cation (Na(+) and Mg(2+)), which forms a bridge with LHA and enhances the intramolecular reaction among these functional groups, therefore inducing the coiling up within the LHA molecule. In addition, the decrease of the K DOC with added MgCl2/MgSO4 (75-95 %) is higher than that with added NaCl/Na2SO4 (20-75 %), indicating that the K DOC was affected by the charge density of cations. The fluorescence intensity of BQH(+) in the absence of LHA (F 0) was found to decay only in the acidic solution with Cl(-), suggesting that Cl(-) might be a heavy atom serving as a quencher in an acidic solution. PMID:25940463

  16. NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions.

    PubMed

    Feeney, James; Birdsall, Berry; Kovalevskaya, Nadezhda V; Smurnyy, Yegor D; Navarro Peran, Emna M; Polshakov, Vladimir I

    2011-05-10

    In order to examine the origins of the large positive cooperativity (ΔG(0)(coop) = -2.9 kcal mol(-1)) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A-B loop region and part of helix B (residues 15-31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to -0.95 kcals mol(-1) if 80% of A-B loop requires refolding). Comparisons of Cα-Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to -1.4 kcal mol(-1)). PMID:21410224

  17. NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

    PubMed Central

    2011-01-01

    In order to examine the origins of the large positive cooperativity (ΔG0coop = −2.9 kcal mol−1) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A−B loop region and part of helix B (residues 15−31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to −0.95 kcals mol−1 if 80% of A−B loop requires refolding). Comparisons of Cα−Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to −1.4 kcal mol−1). PMID:21410224

  18. 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

    ERIC Educational Resources Information Center

    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…

  19. 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.

    PubMed

    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.

  20. Enzyme dynamics from NMR spectroscopy.

    PubMed

    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

  1. Enzyme Dynamics from NMR Spectroscopy

    PubMed Central

    2016-01-01

    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

  2. NMR-based analysis of aminoglycoside recognition by the resistance enzyme ANT(4'): the pattern of OH/NH3(+) substitution determines the preferred antibiotic binding mode and is critical for drug inactivation.

    PubMed

    Revuelta, Julia; Vacas, Tatiana; Torrado, Mario; Corzana, Francisco; Gonzalez, Carlos; Jiménez-Barbero, Jesús; Menendez, Margarita; Bastida, Agatha; Asensio, Juan Luis

    2008-04-16

    The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside

  3. Molecular-Level Examination of Cu2+ Binding Structure for Amyloid Fibrils of 40-Residue Alzheimer’s β by Solid-State NMR Spectroscopy

    PubMed Central

    Parthasarathy, Sudhakar; Long, Fei; Miller, Yifat; Xiao, Yiling; McElheny, Dan; Thurber, Kent; Ma, Buyong; Nussinov, Ruth; Ishii, Yoshitaka

    2011-01-01

    Cu2+ binding to Alzheimer’s β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer’s senile plaque and such association of Aβ with Cu2+ triggers the production of neurotoxic reactive oxygen species (ROS) such as H2O2. However, detailed binding sites and binding structures of Cu2+ to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu2+ binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution 13C SSNMR for full-length 40-residue Aβ(1–40). Selective quenching observed in 13C SSNMR of Cu2+-bound Aβ(1–40) suggested that primary Cu2+ binding sites in Aβ(1–40) fibrils include Nε in His-13 and His-14, and carboxyl groups in Val-40 as well as in Glu side chains (Glu-3, Glu-11, and/or Glu-22). 13C chemical shift analysis demonstrated no major structural changes upon Cu2+ binding in the hydrophobic core regions (residues 18–25 and 30–36). Although the ROS production via oxidization of Met-35 in the presence of Cu2+ has been long suspected, our SSNMR analysis of 13CεH3-S- in M35 showed little changes after Cu2+ binding, excluding the possibility of Met-35 oxidization by Cu2+ alone. Preliminary molecular dynamics (MD) simulations on Cu2+-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu2+-binding modes unique in Aβ fibril, which are realized by both intra- and inter-molecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements. PMID:21341665

  4. Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47phox Phox Homology (PX) Domain Revealed by NMR*

    PubMed Central

    Stampoulis, Pavlos; Ueda, Takumi; Matsumoto, Masahiko; Terasawa, Hiroaki; Miyano, Kei; Sumimoto, Hideki; Shimada, Ichio

    2012-01-01

    The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47phox PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P2 binding mode of the p47phox PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P2-binding site, which includes the residues of helices α1 and α1′ and the following loop up to the distorted left-handed PPII helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47phox PX domain suggest that different types of phosphoinositides sequentially bind to the p47phox PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase. PMID:22493288

  5. Are Fundamental Constants Really Constant?

    ERIC Educational Resources Information Center

    Swetman, T. P.

    1972-01-01

    Dirac's classical conclusions, that the values of e2, M and m are constants and the quantity of G decreases with time. Evoked considerable interest among researchers and traces historical development by which further experimental evidence points out that both e and G are constant values. (PS)

  6. Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation.

    PubMed

    Ma, Xiaoli; He, Jiawei; Yan, Jin; Wang, Qing; Li, Hui

    2016-03-25

    Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy. PMID:26892221

  7. Efficient Atomistic Simulation of Pathways and Calculation of Rate Constants for a Protein-Peptide Binding Process: Application to the MDM2 Protein and an Intrinsically Disordered p53 Peptide.

    PubMed

    Zwier, Matthew C; Pratt, Adam J; Adelman, Joshua L; Kaus, Joseph W; Zuckerman, Daniel M; Chong, Lillian T

    2016-09-01

    The characterization of protein binding processes - with all of the key conformational changes - has been a grand challenge in the field of biophysics. Here, we have used the weighted ensemble path sampling strategy to orchestrate molecular dynamics simulations, yielding atomistic views of protein-peptide binding pathways involving the MDM2 oncoprotein and an intrinsically disordered p53 peptide. A total of 182 independent, continuous binding pathways were generated, yielding a kon that is in good agreement with experiment. These pathways were generated in 15 days using 3500 cores of a supercomputer, substantially faster than would be possible with "brute force" simulations. Many of these pathways involve the anchoring of p53 residue F19 into the MDM2 binding cleft when forming the metastable encounter complex, indicating that F19 may be a kinetically important residue. Our study demonstrates that it is now practical to generate pathways and calculate rate constants for protein binding processes using atomistic simulation on typical computing resources. PMID:27532687

  8. NMR Studies of Peroxidases.

    NASA Astrophysics Data System (ADS)

    Veitch, Nigel Charles

    Available from UMI in association with The British Library. Requires signed TDF. Peroxidases are a haem-containing group of enzymes with a wide diversity of function within biological systems. While a common characteristic is the ability to catalyse the conversion of hydrogen peroxide to water, it is the accompanying processes of hormone synthesis and degradation which have generated such a high level of interest. However, information at the molecular level is limited to a single well-resolved crystal structure, that of yeast cytochrome c peroxidase. This thesis presents a strategy for the investigation of peroxidase structure and function based on proton nuclear magnetic resonance spectroscopy, a technique which has the ability to address aspects of both protein structure and protein dynamics in solution. The application of one- and two-dimensional NMR techniques has been developed in the context of plant peroxidases, notably the isoenzyme HRP-C derived from the horseradish root. Characterisation of the proton NMR spectra of HRP -C in resting and ligated states provided new information enabling the structure of the binding site for aromatic donor molecules, such as indole-3-propionic, ferulic and benzhydroxamic acids, to be resolved. In order to overcome difficulties encountered with a protein of the complexity of peroxidase, additional information was obtained from chemical shift parameters and the use of peroxidase variants produced by site-directed mutagenesis. A comparative study using NMR spectroscopy was undertaken for wild-type recombinant HRP-C expressed in Escherichia coli, and two protein variants with substitutions made to residues located on the distal side of the haem pocket, Phe41 to Val and Arg38 to Lys. NMR analyses of a plant peroxidase from barley grains and the fungal peroxidase from Coprinus cinereus were also successful using methods conceived with HRP-C. Examination of three specifically constructed recombinant protein variants of C. cinereus

  9. Dataset of the binding kinetic rate constants of anti-PCSK9 antibodies obtained using the Biacore T100, ProteOn XPR36, Octet RED384, and IBIS MX96 biosensor platforms.

    PubMed

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2016-09-01

    Here we provide data from a head-to-head comparison study using four biosensor platforms: GE Healthcare׳s Biacore T100, Bio-Rad׳s ProteOn XPR36, ForteBio׳s Octet RED384, and Wasatch Microfluidics׳s IBIS MX96. We used these instruments to analyze the binding interactions of a panel of ten high-affinity monoclonal antibodies with their antigen, human proprotein convertase subtilisin kexin type 9 (PCSK9). For each instrument, binding curves obtained at multiple densities of surface antibodies were fit to the 1:1 Langmuir kinetic model, and the association and dissociation rate constants and corresponding affinity constants were calculated. The data supplied in this article accompany the research article entitled, "Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics" (Yang et al., 2016) [1], which further discusses the strengths and weaknesses of each biosensor platform with an emphasis on data consistency, comparability, and operational efficiency. PMID:27547794

  10. Deuterium Exchange Kinetics by NMR.

    ERIC Educational Resources Information Center

    Roper, G. C.

    1985-01-01

    Describes a physical chemistry experiment which allows such concepts as kinetics, catalysis, isotope shifts, coupling constants, and the use of nuclear magnetic resonance (NMR) for quantitative work to be covered in the same exercise. Background information, experimental procedures used, and typical results obtained are included. (JN)

  11. Characterization of the interaction constants for the binding of two unlabelled ligands to the sites of a receptor. An experimental strategy.

    PubMed

    Swillens, S; Gourdin, A; Delahaut, O

    1989-05-01

    The model describing the binding of three ligands to the sites of a receptor is theoretically studied in order to characterize the binding interaction between two unlabelled ligands. For this, the binding of a third labelled ligand, the affinity of which is dependent on the presence of the unlabelled ligands, must be measured in different conditions. This paper describes an experimental strategy leading to an accurate determination of the equilibrium parameters of the model. This strategy, which assumes that the model is compatible with the data, rests on the determination of the optimal experimental conditions, i.e. the choice of the ligand concentrations which minimize the error of the parameter estimates. For this purpose, a computer program which runs on a microcomputer under the MS-DOS operating system has been elaborated on the basis of the D-optimization criterion. This work shows that such a computer analysis is essential to the determination of the optimal experimental design, even when very simple biochemical systems are considered. PMID:2770883

  12. 1H NMR Study of the Enantioselective Binding of λ- and Δ-[Ru(bpy)2(m-bpy-GHK)]Cl2 to the Deoxynucleotide Duplex d(5'-C1G2C3G4A5A6T7T8C9G10C11G12-3')2

    PubMed Central

    Myari, A.; Garoufis, A.

    2005-01-01

    The interaction of the diastereomeric complexes Λ- and Δ - [Ru(bpy)2(m - GHK)]Cl2, (GHK = glycine-histidine-lysine) to the deoxynucleotide duplex d(5'-CGCGAATTCGCG-3')2 was studied by means of 1H NMR spectroscopy. The diastereomers interact with the oligonucleotide duplex differently. The Δ - [Ru(bpy)2 (m - GHK)]Cl2 is characterized by major groove binding close to the central part of the oligonucleotide, with both the peptide and the bipyridine ligand of the complex involved in the binding. The λ - [Ru(bpy)2 (m - bpy - GHK)]C2 binds loosely, approaching the helix from the minor groove. The NMR analysis shows that the peptide (GHK) binding has a determinative role in the interactions of both diastereomers with the oligonucleotide. PMID:18365093

  13. The investigation of membrane binding by amphibian peptide agonists of CCK2R using (31)P and (2)H solid-state NMR.

    PubMed

    Sherman, Patrick J; Separovic, Frances; Bowie, John H

    2014-05-01

    It has been proposed that some neuropeptides may be anchored to the cell membranes prior to attaching to the adjacent active sites of transmembrane receptors. The three amphibian skin neuropeptides signiferin 1 [RLCIPYIIPC(OH)] (smooth muscle active and immunomodulator), riparin 1.1 [[RLCIPVIFPC(OH)] (immunomodulator) and rothein 1 [SVSNIPESIGF(OH)] (immunomodulator) act via CCK2 transmembrane receptors. A combination of (31)P and (2)H solid state NMR studies of each of these three peptides in eukaryotic phospholipid models at 25°C shows that rothein 1 does not interact with the membrane at all. In contrast, both of the cyclic disulfides signiferin 1 and riparin 1.1 interact with phospholipid head groups and partially penetrate into the upper leaflet of the model bilayer, but to different extents. These interactions are not sufficiently effective to cause disruption of the lipid bilayer since the peptides are not antimicrobial, anticancer, antifungal nor active against enveloped viruses. PMID:24582625

  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.

    PubMed

    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

    SciTech Connect

    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. Disentangling scalar coupling patterns by real-time SERF NMR.

    PubMed

    Gubensäk, Nina; Fabian, Walter M F; Zangger, Klaus

    2014-10-21

    Scalar coupling constants and signal splitting patterns in NMR spectra contain a wealth of short-range structural information. The extraction of these parameters from (1)H NMR spectra is often prohibited by simultaneous scalar coupling interactions with several other protons. Here we present a high-resolution NMR experiment where scalar coupling to only one selected signal is visible. All other couplings are removed from the spectrum. This real-time selectively refocused NMR experiment is achieved by spatially selective homonuclear broadband decoupling combined with selective refocusing during acquisition. It allows the unperturbed extraction of scalar coupling constants from the highly resolved acquisition dimension of NMR spectra.

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

    USGS Publications Warehouse

    Leenheer, J.A.; Brown, G.K.; MacCarthy, P.; Cabaniss, S.E.

    1998-01-01

    Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ 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 Ca2+ 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 13C NMR, 1H NMR, and FT-1R 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 Ca2+ 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.Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ 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 Ca2+ 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 13C NMR, 1H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that

  18. Application of high resolution NMR, ESR, and gamma-ray scintillation spectroscopy to the study of ligand binding in proteins. [Torpedo californica

    SciTech Connect

    Lancione, G.V.

    1982-01-01

    Electron spin resonance spectroscopy has been employed to study the nature of the ligand binding site of alpha-1-antitrypsin. Spectra of spin-labeled alpha-1-antitrypsin were recorded at pH's ranging from 2.4 to 12.5. This data demonstrates the tight binding of the spin-label to the protease, and the sensitivity of the bound spin-label to informational changes in the protease inhibitor. A molecular dipstick approach has also been applied to this system and has yielded information on the geometry of the cleft accommodating the spin-label. /sup 160/Terbium(III) exchange experiments have been performed on the acetylcholine receptor protein isolated from Torpedo californica, employing a specially designed flow dialysis apparatus constructed in the laboratory. The apparatus is designed to allow continuous monitoring of /sup 160/Tb(III) gamma-ray emission from the protein compartment of the flow dialysis cell. Nicotinic ligand-induced displacement of /sup 160/Tb(III) from the nicotinic binding site of the receptor was monitored as a funtion of (1) the concentration of nicotinic ligand in the washout buffer, and (2) the nature of the nicotinic ligand in the buffer. Measured /sup 160/Tb(III) exchange half-lives indicate (1) a direct relationship between /sup 160/Tb(III) displacement and nicotinic ligand concentration in the wash-out buffer, and (2) an enhanced /sup 160/Tb(III) displacement for nicotinic agents possessing quaternary ammonium functions.

  19. Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy.

    PubMed

    Omichinski, J G; Clore, G M; Sakaguchi, K; Appella, E; Gronenborn, A M

    1991-11-01

    A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein. PMID:1959614

  20. Exploring Weak Ligand–Protein Interactions by Long-Lived NMR States: Improved Contrast in Fragment-Based Drug Screening**

    PubMed Central

    Buratto, Roberto; Mammoli, Daniele; Chiarparin, Elisabetta; Williams, Glyn; Bodenhausen, Geoffrey

    2014-01-01

    Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants KD. This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods. PMID:25196717

  1. 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.

    PubMed

    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.

  2. Bicyclic and acyclic diamides: comparison of their aqueous phase binding constants with Nd(III), Am(III), Pu(IV), Np(V), Pu(VI), and U(VI).

    PubMed

    Sinkov, Serguei I; Rapko, Brian M; Lumetta, Gregg J; Hay, Benjamin P; Hutchison, James E; Parks, Bevin W

    2004-12-27

    This report describes affinity measurements for two, water-soluble, methyl-alkylated diamides incorporating the malonamide functionality, N,N,N',N' tetramethylmalonamide (TMMA) and a bicyclic diamide (1a), toward actinide metal cations (An) in acidic nitrate solutions. Ligand complexation to actinides possessing oxidation states ranging from +3 to +6 was monitored through optical absorbance spectroscopy, and formation constants were obtained from the refinement of the spectrophotometric titration data sets. Species analysis gives evidence for the formation of 1, 4, 1, and 2 spectrophotometrically observable complexes by TMMA to An(III, IV, V, and VI), respectively, while for 1a, the respective numbers are 3, 4, 2, and 2. Consistent with the preorganization of 1a toward actinide binding, a significant difference is found in the magnitudes of their respective formation constants at each complexation step. It has been found that the binding affinity for TMMA follows the well-established order An(V) < An(III) < An(VI) < An(IV). However, with 1a, Np(V) forms stronger complexes than Am(III). The complexation of 1a with Np(V) and Pu(VI) at an acidity of 1.0 M is followed by reduction to Np(IV) and Pu(IV), whereas TMMA does not perturb the initial oxidation state for these dioxocations. These measurements of diamide binding affinity mark the first time single-component optical absorbance spectra have been reported for a span of actinide-diamide complexes covering all common oxidation states in aqueous solution.

  3. Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.

    PubMed

    Shadle, S E; Allen, D F; Guo, H; Pogozelski, W K; Bashkin, J S; Tullius, T D

    1997-02-15

    A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

  4. Exploring Chromophore-Binding Pocket: High-Resolution Solid-State H-C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme.

    PubMed

    Song, Chen; Lang, Christina; Mailliet, Jo; Hughes, Jon; Gärtner, Wolfgang; Matysik, Jörg

    2012-02-01

    High-resolution two-dimensional (2D) (1)H-(13)C heteronuclear correlation spectra are recorded for selective observation of interfacial 3-5.5 Å contacts of the uniformly (13)C-labeled phycocyanobilin (PCB) chromophore with its unlabeled binding pocket. The experiment is based on a medium- and long-distance heteronuclear correlation (MELODI-HETCOR) method. For improving (1)H spectral resolution, a windowed phase-modulated Lee-Goldburg (wPMLG) decoupling scheme is applied during the t(1) evolution period. Our approach allows for identification of chromophore-protein interactions, in particular for elucidation of the hydrogen-bonding networks and charge distributions within the chromophore-binding pocket. The resulting pulse sequence is tested on the cyanobacterial (Cph1) phytochrome sensory module (residues 1-514, Cph1Δ2) containing uniformly (13)C- and (15)N-labeled PCB chromophore (u-[(13)C,(15)N]-PCB-Cph1Δ2) at 17.6 T. PMID:22303079

  5. Solution NMR conformation of glycosaminoglycans.

    PubMed

    Pomin, Vitor H

    2014-04-01

    Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structural, dynamical, conformational and intermolecular binding aspects of carbohydrates. Particularly in the field of conformation, NOE resonances, scalar couplings, residual dipolar couplings, and chemical shift anisotropy offsets have been the principal NMR parameters utilized. Molecular dynamics calculations restrained by NMR-data input are usually employed in conjunction to generate glycosidic bond dihedral angles. Glycosaminoglycans (GAGs) are a special class of sulfated polysaccharides extensively studied worldwide. Besides regulating innumerous physiological processes, these glycans are also widely explored in the global market as either clinical or nutraceutical agents. The conformational aspects of GAGs are key regulators to the quality of interactions with the functional proteins involved in biological events. This report discusses the solution conformation of each GAG type analyzed by one or more of the above-mentioned methods.

  6. Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

    PubMed Central

    Behling, R W; Yamane, T; Navon, G; Sammon, M J; Jelinski, L W

    1988-01-01

    A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state. Images FIGURE 1 PMID:3395661

  7. Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.

    PubMed

    Perry, A; Lian, L Y; Scrutton, N S

    2001-02-15

    A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin. PMID

  8. Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.

    PubMed Central

    Perry, A; Lian, L Y; Scrutton, N S

    2001-01-01

    A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin. PMID

  9. 7Li NMR study of normal human erythrocytes

    NASA Astrophysics Data System (ADS)

    Pettegrew, J. W.; Post, J. F. M.; Panchalingam, K.; Withers, G.; Woessner, D. E.

    The biological action of lithium is of great interest because of the therapeutic efficacy of the cation in manic-depressive illness. To investigate possible molecular interactions of lithium, 7Li NMR studies were conducted on normal human erythrocytes which had been incubated with lithium chloride. The uptake of lithium ions was followed by 7Li NMR, using a dysprosium, tripolyphosphate shift reagent. Lithium uptake followed single-exponential kinetics with a time constant of 14.7 h. The intracellular lithium relaxation times were T 1 ⋍ 5 s and T 2 ⋍ 0.15 s, which implies a lengthening of the lithium correlation time. It was found that lithium does not interact significantly with hemoglobin, the erythrocyte membrane, or artificial phospholipid membranes. Based on measurements of lithium T1 and T2 in concentrated agar gels, the large difference between T1 and T2 for intracellular lithium ions may be due to diffusion of the hydrated lithium ion through heterogeneous electrostatic field gradients created by the erythrocyte membrane-associated cytoskeletal network. Lithium binding to the membrane-associated cytoskeleton, however, cannot be ruled out. Because of the large differences between T1 and T2 of intracellular lithium ions, 1Li NMR may be a sensitive and promising noninvasive method to probe the intracellular environment.

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

    PubMed

    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.

  11. Synthesis, spectroscopy, and binding constants of ketocatechol-containing iminodiacetic acid and its Fe(III), Cu(II), and Zn(II) complexes and reaction of Cu(II) complex with H₂O₂ in aqueous solution.

    PubMed

    Gao, Jiaojiao; Xing, Feifei; Bai, Yueling; Zhu, Shourong

    2014-06-01

    A new neuromelanin-like ketocatechol-containing iminodiacetic acid ligand, (N-(3,4-dihydroxyl)phenacylimino)diacetic acid (H4L), which is also quite similar to compounds found in insect cuticle, has been synthesized and characterized. The X-ray crystal structure of H4L has been successfully determined. Proton binding and coordination with Fe(III), Cu(II), and Zn(II) have been studied by potentiometric titrations and UV-vis spectrophotometry in aqueous solution. UV spectra of H4L in the absence and presence of different metal ions indicate complexes formed with the catechol moiety of H4L in aqueous solution. Visible spectra and NMR reveal that H4L with Fe(III), Cu(II), and Zn(II) can all give stable mono-(ML) and dinuclear complexes [M(ML)]. Fe(III) can also form {Fe(FeL)2} and {Fe(FeL)3} species with sufficient base. The process is accompanied by a drastic color change from light blue to deep-blue to wine-red. The Fe(III)-Cu(II) heteronuclear complex also exists in aqueous solution whose spectra are similar to the homonuclear Fe(III) complex. However, the spectra of {Fe(CuL)} shifted to a longer wavelength and {Fe(CuL)2} and {Fe(CuL)3} shifted to a shorter wavelength. Keto-enol tautomerism was observed in weak basic aqueous solution as indicated by (1)H NMR spectra. The reaction products of Cu(II) complex with H2O2 depend on the H2O2 concentration and pH value. Low concentrations of H2O2 oxidize H4L to a series of semiquinone and quinone compounds with absorption maxima at 314-400 nm, while a high concentration of H2O2 oxidizes H4L to colorless muconic acid derivatives. NaIO4 gives different oxidase products, but no 2,4,5-trihydroxyphenylalanine quinone (TPQ)-like hydroxyquinone can be found.

  12. Synthesis and structural analysis using 2-D NMR of Sialyl Lewis X (SLe{sup x}) and Lewis X (Le{sup x}) oligosaccharides: Ligands related to E-selectin [ELAM-1] binding

    SciTech Connect

    Ball, G.E.; Nagy, J.O.; Brown, E.G.

    1992-06-17

    The sialyl Lewis X (SLe{sup x}) determinant (NeuAc-{alpha}-2,3-Gal-{beta}-1,4-[Fuc-{alpha}-1,3]-GlcNAc), compound 1, is a ligand for E-selectin (endothelial leucocyte adhesion molecule 1, or ELAM-1), a member of the selectin family of cell adhesion molecules. Interactions between E-selectin and leucocyte-bound SLe{sup x} or closely related oligosaccharides are thought to be important early events in the inflammation process. Binding analysis has shown that the sialic acid (NeuAc) and the fucose (Fuc) moieties are essential for high affinity. The related desialylated trisaccharide Le{sup x} (Gas-{beta}-1,4-[Fuc-{alpha}-1,3]-GlcNAc), for example, is not a high-affinity ligand for E-selectin. In this communication, the authors describe the syntheses of SLe{sup x} 1 and the {beta}-O-allyl glycoside of Le{sup x} 2 using a cloned fucosyltransferase and their complete NMR spectral assignments including ROESY and NOESY experiments in order to investigate the conformation of these compounds in solution. 25 refs., 2 figs.

  13. 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.

    PubMed

    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.

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

    SciTech Connect

    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.

  15. Saturation transfer difference NMR for fragment screening.

    PubMed

    Begley, Darren W; Moen, Spencer O; Pierce, Phillip G; Zartler, Edward R

    2013-01-01

    Fragment screening by saturation transfer difference nuclear magnetic resonance (STD-NMR) is a robust method for identifying small molecule binders and is well suited to a broad set of biological targets. STD-NMR is exquisitely sensitive for detecting weakly binding compounds (a common characteristic of fragments), which is a crucial step in finding promising compounds for a fragment-based drug discovery campaign. This protocol describes the development of a library suitable for STD-NMR fragment screening, as well as preparation of protein samples, optimization of experimental conditions, and procedures for data collection and analysis. PMID:24391096

  16. A unique binding cavity for divalent cations in the DNA-metal-chromomycin A3 complex.

    PubMed

    Itzhaki, L; Weinberger, S; Livnah, N; Berman, E

    1990-02-15

    Binding of chromomycin A3 (CRA) to calf thymus DNA was investigated in the presence of divalent cations using visible absorption and 1H-nmr spectroscopies. An apparent equilibrium binding constant (approximately 10(11) M-1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA-M-CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H-nmr spectrum of the above complex using the paramagnetic Co2+ cation, indicating that the metal occupies a unique binding site. Since no induced 1H-nmr chemical shifts were observed for the drug-Co2+ mixture, it was concluded that no metal-drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (less than 0.85 A) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA-M-CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. NMR investigations of molecular dynamics

    NASA Astrophysics Data System (ADS)

    Palmer, Arthur

    2011-03-01

    NMR spectroscopy is a powerful experimental approach for characterizing protein conformational dynamics on multiple time scales. The insights obtained from NMR studies are complemented and by molecular dynamics (MD) simulations, which provide full atomistic details of protein dynamics. Homologous mesophilic (E. coli) and thermophilic (T. thermophilus) ribonuclease H (RNase H) enzymes serve to illustrate how changes in protein sequence and structure that affect conformational dynamic processes can be monitored and characterized by joint analysis of NMR spectroscopy and MD simulations. A Gly residue inserted within a putative hinge between helices B and C is conserved among thermophilic RNases H, but absent in mesophilic RNases H. Experimental spin relaxation measurements show that the dynamic properties of T. thermophilus RNase H are recapitulated in E. coli RNase H by insertion of a Gly residue between helices B and C. Additional specific intramolecular interactions that modulate backbone and sidechain dynamical properties of the Gly-rich loop and of the conserved Trp residue flanking the Gly insertion site have been identified using MD simulations and subsequently confirmed by NMR spin relaxation measurements. These results emphasize the importance of hydrogen bonds and local steric interactions in restricting conformational fluctuations, and the absence of such interactions in allowing conformational adaptation to substrate binding.

  19. Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.

    PubMed

    South, T L; Summers, M F

    1993-01-01

    The nucleic acid interactive properties of a synthetic peptide with sequence of the N-terminal CCHC zinc finger (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence-dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3' side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding to a portion of the HIV-1 psi-packaging signal, d(ACGCC), forms a relatively tight complex with Zn(HIV1-F1) (Kd = 5 x 10(-6) M). Two-dimensional nuclear Overhauser effect (NOESY) data indicate that the bound nucleic acid exists predominantly in a single-stranded, A-helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three-dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site-directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.

  20. Substituent effects on fluoride binding by lanthanide complexes of DOTA-tetraamides.

    PubMed

    Blackburn, Octavia A; Routledge, Jack D; Jennings, Laura B; Rees, Nicholas H; Kenwright, Alan M; Beer, Paul D; Faulkner, Stephen

    2016-02-21

    Fluoride binding by a series of europium and ytterbium complexes of DOTA-tetraamide ligands derived from primary, secondary and tertiary amides has been studied by NMR and luminescence spectroscopies. In all the systems studied, fluoride binding results in a change in the nature of the magnetic anisotropy at the metal centre from an easy axis, to an easy plane anisotropy. This results in reversal of the peaks in the NMR spectra, and in changes to the fine structure of the luminescence spectra. Furthermore, changes to the periphery of the binding cavity are implicated in determining the affinity constant for fluoride. There are clear differences in the entropic contribution to the free energy of activation between systems with benzylic amides and those with methylamides. PMID:26765788

  1. 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

    NASA Astrophysics Data System (ADS)

    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.

  2. NMR of group 2 element quadrupolar nuclei and some applications in materials science and biology

    NASA Astrophysics Data System (ADS)

    Li, Xiaohua

    1999-11-01

    fundamental coordination chemistry of strontium present in the sol, the homogeneity of the ceramic was substantially enhanced, and the resulting electronic properties were improved. In another application of the new technique which have been developed, 87Sr NMR was employed to study the metal ion binding properties of alpha-Lactalbumin. The NMR data was fitted into a model with one high affinity and one low affinity site in alpha-Lactalbumin. The calculation of the correlation time, quadrupolar coupling constant provided the opportunity to study the location and the structure of the binding sites. In the application of the new technique in the area of biology, 25Mg NMR was employed to study the metal ion binding properties of bacteriorhodopsin. We presented the results of our studies regarding to two important questions in the photo conversion process: (1) The number of metal binding sites and their binding constants; and (2) What is the role of metal cations in the proton dissociation process/proton transfer (e.g., the key issue of the switch of the proton pump)? 113Cd NM of metal-bR complexes was employed as a probe of the chemical structure of bR calcium binding sites. We discuss the results in terms of the possible involvement of the metal cations in the bR function.

  3. NMR Logging to Estimate Hydraulic Conductivity in Unconsolidated Aquifers.

    PubMed

    Knight, Rosemary; Walsh, David O; Butler, James J; Grunewald, Elliot; Liu, Gaisheng; Parsekian, Andrew D; Reboulet, Edward C; Knobbe, Steve; Barrows, Mercer

    2016-01-01

    Nuclear magnetic resonance (NMR) logging provides a new means of estimating the hydraulic conductivity (K) of unconsolidated aquifers. The estimation of K from the measured NMR parameters can be performed using the Schlumberger-Doll Research (SDR) equation, which is based on the Kozeny-Carman equation and initially developed for obtaining permeability from NMR logging in petroleum reservoirs. The SDR equation includes empirically determined constants. Decades of research for petroleum applications have resulted in standard values for these constants that can provide accurate estimates of permeability in consolidated formations. The question we asked: Can standard values for the constants be defined for hydrogeologic applications that would yield accurate estimates of K in unconsolidated aquifers? Working at 10 locations at three field sites in Kansas and Washington, USA, we acquired NMR and K data using direct-push methods over a 10- to 20-m depth interval in the shallow subsurface. Analysis of pairs of NMR and K data revealed that we could dramatically improve K estimates by replacing the standard petroleum constants with new constants, optimal for estimating K in the unconsolidated materials at the field sites. Most significant was the finding that there was little change in the SDR constants between sites. This suggests that we can define a new set of constants that can be used to obtain high resolution, cost-effective estimates of K from NMR logging in unconsolidated aquifers. This significant result has the potential to change dramatically the approach to determining K for hydrogeologic applications. PMID:25810149

  4. NMR Logging to Estimate Hydraulic Conductivity in Unconsolidated Aquifers.

    PubMed

    Knight, Rosemary; Walsh, David O; Butler, James J; Grunewald, Elliot; Liu, Gaisheng; Parsekian, Andrew D; Reboulet, Edward C; Knobbe, Steve; Barrows, Mercer

    2016-01-01

    Nuclear magnetic resonance (NMR) logging provides a new means of estimating the hydraulic conductivity (K) of unconsolidated aquifers. The estimation of K from the measured NMR parameters can be performed using the Schlumberger-Doll Research (SDR) equation, which is based on the Kozeny-Carman equation and initially developed for obtaining permeability from NMR logging in petroleum reservoirs. The SDR equation includes empirically determined constants. Decades of research for petroleum applications have resulted in standard values for these constants that can provide accurate estimates of permeability in consolidated formations. The question we asked: Can standard values for the constants be defined for hydrogeologic applications that would yield accurate estimates of K in unconsolidated aquifers? Working at 10 locations at three field sites in Kansas and Washington, USA, we acquired NMR and K data using direct-push methods over a 10- to 20-m depth interval in the shallow subsurface. Analysis of pairs of NMR and K data revealed that we could dramatically improve K estimates by replacing the standard petroleum constants with new constants, optimal for estimating K in the unconsolidated materials at the field sites. Most significant was the finding that there was little change in the SDR constants between sites. This suggests that we can define a new set of constants that can be used to obtain high resolution, cost-effective estimates of K from NMR logging in unconsolidated aquifers. This significant result has the potential to change dramatically the approach to determining K for hydrogeologic applications.

  5. NMR analysis of biodiesel

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biodiesel is usually analyzed by the various methods called for in standards such as ASTM D6751 and EN 14214. Nuclear magnetic resonance (NMR) is not one of these methods. However, NMR, with 1H-NMR commonly applied, can be useful in a variety of applications related to biodiesel. These include monit...

  6. Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

    PubMed

    Kucharska, Iga; Liang, Binyong; Ursini, Nicholas; Tamm, Lukas K

    2016-09-13

    Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa. PMID:27532487

  7. Quantitative evaluation of noncovalent interactions between glyphosate and dissolved humic substances by NMR spectroscopy.

    PubMed

    Mazzei, Pierluigi; Piccolo, Alessandro

    2012-06-01

    Interactions of glyphosate (N-phosphonomethylglycine) herbicide (GLY) with soluble fulvic acids (FAs) and humic acids (HAs) at pH 5.2 and 7 were studied by (1)H and (31)P NMR spectroscopy. Increasing concentrations of soluble humic matter determined broadening and chemical shift drifts of proton and phosphorus GLY signals, thereby indicating the occurrence of weak interactions between GLY and humic superstructures. Binding was larger for FAs and pH 5.2 than for HAs and pH 7, thus suggesting formation of hydrogen bonds between GLY carboxyl and phosphonate groups and protonated oxygen functions in humic matter. Changes in relaxation and correlation times of (1)H and (31)P signals and saturation transfer difference NMR experiments confirmed the noncovalent nature of GLY-humic interactions. Diffusion-ordered NMR spectra allowed calculation of the glyphosate fraction bound to humic superstructures and association constants (K(a)) and Gibbs free energies of transfer for GLY-humic complex formation at both pH values. These values showed that noncovalent interactions occurred most effectively with FAs and at pH 5.2. Our findings indicated that glyphosate may spontaneously and significantly bind to soluble humic matter by noncovalent interactions at slightly acidic pH and, thus, potentially pollute natural water bodies by moving through soil profiles in complexes with dissolved humus. PMID:22591574

  8. Applications of high resolution /sup 3/H NMR spectroscopy

    SciTech Connect

    Williams, P.G.

    1987-10-01

    The advantages of tritium as an NMR nucleus are pointed out. Examples of its use are given, including labelled toluene, hydrogenation of ..beta..-methylstyrene, and maltose and its binding proteins. 7 refs., 2 figs. (DLC)

  9. NMR study of chloride ion interactions with thylakoid membranes

    PubMed Central

    Baianu, I. C.; Critchley, C.; Govindjee; Gutowsky, H. S.

    1984-01-01

    The role of Cl- in photosynthetic O2 evolution has been investigated by observing the 35Cl NMR linewidth under a variety of conditions in aqueous suspensions of chloroplasts, primarily for the halophytes Avicennia germinans, Avicennia marina, and Aster tripolium but also for spinach. The line broadening shows there is weak, ionic binding of Cl- to thylakoids, the bound Cl- exchanging rapidly (>>104 sec-1) with free Cl- in solution. The binding is necessary for O2 evolution to occur. Michaelis-Menten constants obtained from the Cl- dependence of the O2 evolution rate are ≈15-70 mM for the halophytes compared with 0.6 mM for spinach (0.5 mM with Br-). There appear to be two types of Cl- binding sites in halophytes, of which the stronger is the activator, at lower [Cl-], of O2 evolution. The 35Cl line broadening includes a nonspecific interaction, which becomes apparent at high Cl- concentrations (≥0.5 M). PMID:16593474

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

    PubMed

    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.

  11. Inclusion complex formation of ternary system: Fluoroscein-p-sulfonato calix[4]arene-Cu(2+) by cooperative binding.

    PubMed

    Gawhale, Sharadchandra; Jadhav, Ankita; Rathod, Nilesh; Malkhede, Dipalee; Chaudhari, Gajanan

    2015-09-01

    The aqueous solution of fluorescein-para sulfonato calix[4]arene-metal ion complex has been studied based on absorption, fluorescence, (1)H NMR and FTIR spectroscopic results. It was found that the fluorescence intensity quenched regularly upon addition of pSCX4 and metal ion. The quenching constants and binding constants were determined for pSCX4-FL and pSCX4-FL-Cu(2+) systems. 1:1 stoichiometry is obtained for pSCX4-Cu(2+) system by continuous variation method. The NMR and IR results indicates the interaction among FL, pSCX4 and Cu(2+). The combined results demonstrate the cooperative binding to design the complex for ternary system. The life time for binary and ternary system has been studied.

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

    PubMed

    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.

  13. A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase.

    PubMed

    Velyvis, Algirdas; Yang, Ying R; Schachman, Howard K; Kay, Lewis E

    2007-05-22

    The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, (1)H,(13)C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L') between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is "invisible" in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.

  14. Polarization transfer NMR imaging

    DOEpatents

    Sillerud, Laurel O.; van Hulsteyn, David B.

    1990-01-01

    A nuclear magnetic resonance (NMR) image is obtained with spatial information modulated by chemical information. The modulation is obtained through polarization transfer from a first element representing the desired chemical, or functional, information, which is covalently bonded and spin-spin coupled with a second element effective to provide the imaging data. First and second rf pulses are provided at first and second frequencies for exciting the imaging and functional elements, with imaging gradients applied therebetween to spatially separate the nuclei response for imaging. The second rf pulse is applied at a time after the first pulse which is the inverse of the spin coupling constant to select the transfer element nuclei which are spin coupled to the functional element nuclei for imaging. In a particular application, compounds such as glucose, lactate, or lactose, can be labeled with .sup.13 C and metabolic processes involving the compounds can be imaged with the sensitivity of .sup.1 H and the selectivity of .sup.13 C.

  15. Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.

    PubMed

    Kim, Yaewon; Hilty, Christian

    2015-04-13

    Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

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

    PubMed

    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. PMID:27132848

  17. Experimental and Theoretical Studies on Halide Binding with a p-Xylyl-Based Azamacrocycle

    PubMed Central

    2015-01-01

    A p-xylyl-based macrocycle L has been synthesized and its binding properties with halides have been investigated by 1H NMR titrations, single crystal X-ray diffraction analysis, and density functional theory (DFT) calculations. As investigated by 1H NMR titrations, the ligand preferentially binds a halide in a 1:2 binding mode, with the association constants (in log K2) of 2.82, 2.70, 2.28, and 2.20 for fluoride, chloride, bromide, and iodide, respectively. The overall binding trend was found to be in the order of fluoride > chloride > bromide > iodide, reflecting that the binding strength correlates with the relative basicity and size of the respective halide. Crystallographic studies indicate that the ligand forms 1:2 complexes with chloride, bromide and iodide. In the chloride complex, the ligand is hexaprotonated and each chloride is held via three NH···Cl– bonds. The ligand is tetraprotonated for the other complexes, where each halide is H-bonded to two secondary ammonium NH+ groups via NH···X– bonds. The results of DFT calculations performed on [H6L]6+ at M062x/6-311G (d,p) level in both gas and solvent phases, suggest that the ligand binds halides with the binding energy in the order of F– > Cl– > Br– > I–, supporting the experimental data obtained from 1H NMR studies. Results from DFT calculations further indicate that a 1:2 binding is energetically more favorable than a 1:1 binding of the ligand. PMID:25517862

  18. Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

    SciTech Connect

    Beschiaschvili, G.; Seelig, J. )

    1990-01-09

    The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.

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

    PubMed

    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.

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

    PubMed

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

    2011-12-01

    (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.

  1. Solution NMR studies of supramolecular complexes of [60]- and [70]fullerenes with mono O-substituted calix[6]arene

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Sumanta; Chattopadhyay, Subrata; Nayak, Sandip K.; Banerjee, Manas

    2005-11-01

    Supramolecular complexation of [60]- and [70]fullerenes with 37-allyl-38,39,40,41,42-pentahydroxy-5,11,17,23,29,35-hexa(4- tert butyl)calix[6]arene ( I) has been studied in CCl 4 medium by NMR spectrometric method. All of the complexes are found to be stable with 1:1 stoichiometry. Formation constants ( K) of the above supramolecular complexes have been determined from systematic variation of NMR chemical shifts of specific protons of I in the presence of [60]- and [70]fullerenes. Trends in the K value suggest that [70]fullerene binds more strongly with I relative to [60]fullerene. Both PM3 and ab initio calculations reveal that the intermolecular interaction in the [70]fullerene/ I complex proceeds through quite deep energy minima.

  2. (19)F-encoded combinatorial libraries: discovery of selective metal binding and catalytic peptoids.

    PubMed

    Pirrung, Michael C; Park, Kaapjoo; Tumey, L Nathan

    2002-01-01

    A (19)F NMR method for encoding of combinatorial libraries has been developed. Aryl fluorides whose chemical shifts are modified by aromatic substituents were prepared and attached to resin support beads that were used in the split-pool synthesis of peptoids. The detection of the (19)F NMR signal of tags derived from a single "big bead" was demonstrated. The library diversity arises from peptoid amines and the cyclic anhydrides used in their acylation. The resulting 90-compound library was examined for metal ion binding, and novel ligands for iron and copper were discovered. Their binding constants were determined to be in the low micromolar range using conventional methods. The library was also examined for autocatalysis of acylation, and a molecule possessing the catalytic triad of serine proteases was deduced.

  3. Lectures on pulsed NMR

    SciTech Connect

    Pines, A.

    1986-09-01

    These lectures discuss some recent developments in pulsed NMR, emphasizing fundamental principles with selected illustrative applications. Major topics covered include multiple-quantum spectroscopy, spin decoupling, the interaction of spins with a quantized field, adiabatic rapid passage, spin temperature and statistics of cross-polarization, coherent averaging, and zero field NMR. 55 figs.

  4. Lectures on pulsed NMR

    SciTech Connect

    Pines, A.

    1988-08-01

    These lectures discuss some recent developments in pulsed NMR, emphasizing fundamental principles with selected illustrative applications. Major topics covered include multiple-quantum spectroscopy, spin decoupling, the interaction of spins with a quantized field, adiabatic rapid passage, spin temperature and statistics of cross-polarization, coherent averaging, and zero field NMR. 32 refs., 56 figs.

  5. NMR logging apparatus

    DOEpatents

    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.

  6. NMR methods in combinatorial chemistry.

    PubMed

    Shapiro, M J; Wareing, J R

    1998-06-01

    The use of NMR spectroscopy in combinatorial chemistry has provided a versatile tool for monitoring combinatorial chemistry reactions and for assessing ligand-receptor interactions. The application of magic angle spinning NMR is widespread and has allowed structure determination to be performed on compounds attached to solid supports. A variety of two-dimensional NMR techniques have been applied to enhance the usability of the magic angle spinning NMR data. New developments for solution NMR analysis include high performance liquid chromatography, NMR, mass spectroscopy and flow NMR. NMR based methods currently being investigated may prove valuable as compound screening tools.

  7. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    PubMed

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology. PMID:24524625

  8. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    PubMed

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

  9. Pure shift NMR.

    PubMed

    Zangger, Klaus

    2015-04-01

    Although scalar-coupling provides important structural information, the resulting signal splittings significantly reduce the resolution of NMR spectra. Limited resolution is a particular problem in proton NMR experiments, resulting in part from the limited proton chemical shift range (∼10 ppm) but even more from the splittings due to scalar coupling to nearby protons. "Pure shift" NMR spectroscopy (also known as broadband homonuclear decoupling) has been developed for disentangling overlapped proton NMR spectra. The resulting spectra are considerably simplified as they consist of single lines, reminiscent of proton-decoupled C-13 spectra at natural abundance, with no multiplet structure. The different approaches to obtaining pure shift spectra are reviewed here and several applications presented. Pure shift spectra are especially useful for highly overlapped proton spectra, as found for example in reaction mixtures, natural products and biomacromolecules.

  10. A Flexible Docking Scheme Efficiently Captures the Energetics of Glycan-Cyanovirin Binding

    PubMed Central

    Bolia, Ashini; Woodrum, Brian W.; Cereda, Angelo; Ruben, Melissa A.; Wang, Xu; Ozkan, S. Banu; Ghirlanda, Giovanna

    2014-01-01

    Cyanovirin-N (CVN), a cyanobacterial lectin, exemplifies a class of antiviral agents that inhibit HIV by binding to the highly glycosylated envelope protein gp120. Here, we investigate the energetics of glycan recognition using a computationally inexpensive flexible docking approach, backbone perturbation docking (BP-Dock). We benchmarked our method using two mutants of CVN: P51G-m4-CVN, which binds dimannose with high affinity through domain B, and CVN(mutDB), in which binding to domain B has been abolished through mutation of five polar residues to small nonpolar side chains. We investigated the energetic contribution of these polar residues along with the additional position 53 by docking dimannose to single-point CVN mutant models. Analysis of the docking simulations indicated that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen-bond network that reverberated throughout the binding cavity. N42A decreased the binding score to a level comparable to that of CVN(mutDB) by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score, similar to P51G-m4-CVN. Experimental characterization of the five mutants by NMR spectroscopy confirmed the binding affinity pattern predicted by BP-Dock. Despite their mostly conserved fold and stability, E41A, E41G, and T57A displayed dissociation constants in the millimolar range. N53S showed a binding constant in the low micromolar range, similar to that observed for P51G-m4-CVN. No binding was observed for N42A. Our results show that BP-Dock is a useful tool for rapidly screening the relative binding affinity pattern of in silico-designed mutants compared with wild-type, supporting its use to design novel mutants with enhanced binding properties. PMID:24606938

  11. Fragment-Based Drug Discovery Using NMR Spectroscopy

    PubMed Central

    Harner, Mary J.; Frank, Andreas O.; Fesik, Stephen W.

    2013-01-01

    Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience. PMID:23686385

  12. Vicinal coupling constants and protein dynamics.

    PubMed

    Hoch, J C; Dobson, C M; Karplus, M

    1985-07-16

    The effects of motional averaging on the analysis of vicinal spin-spin coupling constants derived from proton NMR studies of proteins have been examined. Trajectories obtained from molecular dynamics simulations of bovine pancreatic trypsin inhibitor and of hen egg white lysozyme were used in conjunction with an expression for the dependence of the coupling constant on the intervening dihedral angle to calculate the time-dependent behavior of the coupling constants. Despite large fluctuations, the time-average values of the coupling constants are not far from those computed for the average structure in the cases where fluctuations occur about a single potential well. The calculated differences show a high correlation with the variation in the magnitude of the fluctuations of individual dihedral angles. For the cases where fluctuations involve multiple sites, large differences are found between the time-average values and the average structure values for the coupling constants. Comparison of the simulation results with the experimental trends suggests that side chains with more than one position are more common in proteins than is inferred from X-ray results. It is concluded that for the main chain, motional effects do not introduce significant errors where vicinal coupling constants are used in structure determinations; however, for side chains, the motional average can alter deductions about the structure. Accurately measured coupling constants are shown to provide information concerning the magnitude of dihedral angle fluctuations.

  13. Evidence That Antibiotics Bind to Human Mitochondrial Ribosomal RNA Has Implications for Aminoglycoside Toxicity.

    PubMed

    Hong, Seoyeon; Harris, Kimberly A; Fanning, Kathryn D; Sarachan, Kathryn L; Frohlich, Kyla M; Agris, Paul F

    2015-07-31

    Aminoglycosides are a well known antibiotic family used to treat bacterial infections in humans and animals, but which can be toxic. By binding to the decoding site of helix44 of the small subunit RNA of the bacterial ribosome, the aminoglycoside antibiotics inhibit protein synthesis, cause misreading, or obstruct peptidyl-tRNA translocation. Although aminoglycosides bind helix69 of the bacterial large subunit RNA as well, little is known about their interaction with the homologous human helix69. To probe the role this binding event plays in toxicity, changes to thermal stability, base stacking, and conformation upon aminoglycoside binding to the human cytoplasmic helix69 were compared with those of the human mitochondrial and Escherichia coli helix69. Surprisingly, binding of gentamicin and kanamycin A to the chemically synthesized terminal hairpins of the human cytoplasmic, human mitochondrial, and E. coli helix69 revealed similar dissociation constants (1.3-1.7 and 4.0-5.4 μM, respectively). In addition, aminoglycoside binding enhanced conformational stability of the human mitochondrial helix69 by increasing base stacking. Proton one-dimensional and two-dimensional NMR suggested significant and specific conformational changes of human mitochondrial and E. coli helix69 upon aminoglycoside binding, as compared with human cytoplasmic helix69. The conformational changes and similar aminoglycoside binding affinities observed for human mitochondrial helix69 and E. coli helix69, as well as the increase in structural stability shown for the former, suggest that this binding event is important to understanding aminoglycoside toxicity. PMID:26060252

  14. NMR imaging microscopy

    SciTech Connect

    Not Available

    1986-10-01

    In the past several years, proton nuclear magnetic resonance (NMR) imaging has become an established technique in diagnostic medicine and biomedical research. Although much of the work in this field has been directed toward development of whole-body imagers, James Aguayo, Stephen Blackband, and Joseph Schoeninger of the Johns Hopkins University School of Medicine working with Markus Hintermann and Mark Mattingly of Bruker Medical Instruments, recently developed a small-bore NMR microscope with sufficient resolution to image a single African clawed toad cell (Nature 1986, 322, 190-91). This improved resolution should lead to increased use of NMR imaging for chemical, as well as biological or physiological, applications. The future of NMR microscopy, like that of many other newly emerging techniques, is ripe with possibilities. Because of its high cost, however, it is likely to remain primarily a research tool for some time. ''It's like having a camera,'' says Smith. ''You've got a way to look at things at very fine levels, and people are going to find lots of uses for it. But it is a very expensive technique - it costs $100,000 to add imaging capability once you have a high-resolution NMR, which itself is at least a $300,000 instrument. If it can answer even a few questions that can't be answered any other way, though, it may be well worth the cost.''

  15. On the Khinchin Constant

    NASA Technical Reports Server (NTRS)

    Bailey, David H.; Borwein, Jonathan M.; Crandall, Richard E.; Craw, James M. (Technical Monitor)

    1995-01-01

    We prove known identities for the Khinchin constant and develop new identities for the more general Hoelder mean limits of continued fractions. Any of these constants can be developed as a rapidly converging series involving values of the Riemann zeta function and rational coefficients. Such identities allow for efficient numerical evaluation of the relevant constants. We present free-parameter, optimizable versions of the identities, and report numerical results.

  16. Binding of acetylcholine and tetramethylammonium to flexible cyclophane receptors: improving on binding ability by optimizing host's geometry.

    PubMed

    Sarri, Paolo; Venturi, Francesca; Cuda, Francesco; Roelens, Stefano

    2004-05-28

    The structure of a cyclophanic tetraester (1), previously employed for investigations on the cation-pi interaction, has been optimized to better accommodate acetylcholine (ACh) and tetramethylammonium (TMA) guests. Following indications from molecular modeling calculations, a flexible cyclophane receptor of significantly improved binding properties has been obtained by removing the four carbonyl groups of the parent host. 2,11,20,29-Tetraoxa[3.3.3.3]paracyclophane (2) was prepared by an improved procedure, which was conveniently devised to avoid the formation of contiguous cyclooligomers that caused serious separation issues. Association of 2 with TMA picrate was measured in CDCl(3) at T = 296 K by (1)H NMR titrations and compared to binding data obtained for a set of reference hosts, including the parent tetraester 1, the corresponding cyclophanic tetraamine, the open-chain counterpart of 2, and its cyclooligomers from pentamer to octamer. Binding enhancements ranging from 15-fold (with respect to the tetraester and the tetraamine) to over 80-fold (with respect to the open-chain tetraether) were achieved by geometry optimization of the host. Binding of 2 to ACh and TMA was investigated for a variety of counterions. A constant binding free energy increment of nearly 8 kJ mol(-1) with respect to 1 was observed, independent from the anion and irrespective of the different structure of the cationic guests. Results showed that the electrostatic inhibiting contribution of the counterion to the cation's binding is a characteristic constant of each anion. The value of -Delta G degrees = 44.9 kJ mol(-1) extrapolated for TMA in the absence of a counterion indicates that 28-34 kJ mol(-1) of binding free energy are lost in ion pairing.

  17. The hubble constant.

    PubMed

    Huchra, J P

    1992-04-17

    The Hubble constant is the constant of proportionality between recession velocity and distance in the expanding universe. It is a fundamental property of cosmology that sets both the scale and the expansion age of the universe. It is determined by measurement of galaxy The Hubble constant is the constant of proportionality between recession velocity and development of new techniques for the measurements of galaxy distances, both calibration uncertainties and debates over systematic errors remain. Current determinations still range over nearly a factor of 2; the higher values favored by most local measurements are not consistent with many theories of the origin of large-scale structure and stellar evolution. PMID:17743107

  18. The cosmological constant

    NASA Technical Reports Server (NTRS)

    Carroll, Sean M.; Press, William H.; Turner, Edwin L.

    1992-01-01

    The cosmological constant problem is examined in the context of both astronomy and physics. Effects of a nonzero cosmological constant are discussed with reference to expansion dynamics, the age of the universe, distance measures, comoving density of objects, growth of linear perturbations, and gravitational lens probabilities. The observational status of the cosmological constant is reviewed, with attention given to the existence of high-redshift objects, age derivation from globular clusters and cosmic nuclear data, dynamical tests of Omega sub Lambda, quasar absorption line statistics, gravitational lensing, and astrophysics of distant objects. Finally, possible solutions to the physicist's cosmological constant problem are examined.

  19. Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigation of the flavin mononucleotide binding site in the reduced and oxidized states

    SciTech Connect

    Stockman, B.J.; Westler, W.M.; Mooberry, E.S.; Markley, J.L.

    1988-01-12

    Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (M/sub r/ approx. 21,000). /sup 1/H, /sup 15/N, and /sup 31/P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched (/sup 15/N) flavodoxin was isolated from Anabaena 7120 grown on K/sup 15/NO/sub 3/ as the sole nitrogen source. /sup 15/N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A /sup 1/H-detected /sup 15/N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.

  20. Dual Species NMR Oscillator

    NASA Astrophysics Data System (ADS)

    Weber, Joshua; Korver, Anna; Thrasher, Daniel; Walker, Thad

    2016-05-01

    We present progress towards a dual species nuclear magnetic oscillator using synchronous spin exchange optical pumping. By applying the bias field as a sequence of alkali 2 π pulses, we generate alkali polarization transverse to the bias field. The alkali polarization is then modulated at the noble gas resonance so that through spin exchange collisions the noble gas becomes polarized. This novel method of NMR suppresses the alkali field frequency shift by at least a factor of 2500 as compared to longitudinal NMR. We will present details of the apparatus and measurements of dual species co-magnetometry using this method. Research supported by the NSF and Northrop-Grumman Corp.

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

    PubMed

    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. PMID:27232663

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

    PubMed

    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.

  3. NMR Studies of Dynamic Biomolecular Conformational Ensembles

    PubMed Central

    Torchia, Dennis A.

    2015-01-01

    Multidimensional heteronuclear NMR approaches can provide nearly complete sequential signal assignments of isotopically enriched biomolecules. The availability of assignments together with measurements of spin relaxation rates, residual spin interactions, J-couplings and chemical shifts provides information at atomic resolution about internal dynamics on timescales ranging from ps to ms, both in solution and in the solid state. However, due to the complexity of biomolecules, it is not possible to extract a unique atomic-resolution description of biomolecular motions even from extensive NMR data when many conformations are sampled on multiple timescales. For this reason, powerful computational approaches are increasingly applied to large NMR data sets to elucidate conformational ensembles sampled by biomolecules. In the past decade, considerable attention has been directed at an important class of biomolecules that function by binding to a wide variety of target molecules. Questions of current interest are: “Does the free biomolecule sample a conformational ensemble that encompasses the conformations found when it binds to various targets; and if so, on what time scale is the ensemble sampled?” This article reviews recent efforts to answer these questions, with a focus on comparing ensembles obtained for the same biomolecules by different investigators. A detailed comparison of results obtained is provided for three biomolecules: ubiquitin, calmodulin and the HIV-1 trans-activation response RNA. PMID:25669739

  4. Design and Nuclear Magnetic Resonance (NMR) Structure Determination of the Second Extracellular Immunoglobulin Tyrosine Kinase A (TrkAIg2) Domain Construct for Binding Site Elucidation in Drug Discovery

    PubMed Central

    2014-01-01

    The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein–ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the 1H–15N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution. PMID:25454499

  5. Fundamental Physical Constants

    National Institute of Standards and Technology Data Gateway

    SRD 121 CODATA Fundamental Physical Constants (Web, free access)   This site, developed in the Physics Laboratory at NIST, addresses three topics: fundamental physical constants, the International System of Units (SI), which is the modern metric system, and expressing the uncertainty of measurement results.

  6. Calculation of magnetostriction constants

    NASA Astrophysics Data System (ADS)

    Tatebayashi, T.; Ohtsuka, S.; Ukai, T.; Mori, N.

    1986-02-01

    The magnetostriction constants h1 and h2 for Ni and Fe metals and the anisotropy constants K1 and K2 for Fe metal are calculated on the basis of the approximate d bands obtained by Deegan's prescription, by using Gilat-Raubenheimer's method. The obtained results are compared with the experimental ones.

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

    PubMed

    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. PMID:25639618

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

    PubMed

    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.

  9. Targeted Molecular Imaging of Cancer Cells Using MS2-Based (129)Xe NMR.

    PubMed

    Jeong, Keunhong; Netirojjanakul, Chawita; Munch, Henrik K; Sun, Jinny; Finbloom, Joel A; Wemmer, David E; Pines, Alexander; Francis, Matthew B

    2016-08-17

    We have synthesized targeted, selective, and highly sensitive (129)Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized (129)Xe NMR signal contrast and hyper-CEST (129)Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized (129)Xe NMR nanoscale biosensor for future applications in cancer detection in vivo. PMID:27454679

  10. The Effect of Mutation of Carboxyl Side-Chain Amino Acids Near the Heme on the Midpoint Potentials and Ligand Binding Constants of Nitrophorin 2 and Its NO, Histamine and Imidazole Complexes

    PubMed Central

    Berry, Robert E.; Shokhirev, Maxim N.; Ho, Arthur Y. W.; Yang, Fei; Shokhireva, Tatiana K.; Zhang, Hongjun; Weichsel, Andrzej; Montfort, William R.; Walker, F. Ann

    2009-01-01

    Nitrophorins (NPs) are a group of NO-carrying heme proteins found in the saliva of a bloodsucking insect from tropical Central and South America, Rhodnius prolixus, the “kissing bug”. NO is kept stable for long periods of time by binding it as an axial ligand to a ferriheme center. The fact that the nitrophorins are stabilized as FeIII-NO proteins is a unique property, for most heme proteins are readily autoreduced by excess NO, and bind NO to the Fe(II) heme irreversibly (Kds in the pM range). In contrast, the nitrophorins, as Fe(III) heme centers, have Kds in the μM to nM range, and thus allow NO to dissociate upon dilution following injection into the tissues of the victim. This NO can cause vasodilation, and thereby allow more blood to be transported to the site of the wound. We have prepared thirteen site-directed mutants of three major nitrophorins, NP2, NP1 and NP4, to investigate the stabilization of the ferric–NO heme center and preservation of reversible binding that facilitates these proteins’ NO storage, transport and release functions. Of the mutations in which Glu and/or Asp were replaced by Ala, most of these carboxyls show a significant role stabilizing FeIII-NO over FeII-NO, with buried E53 of NP2 or E55 of NP1 and NP4 being the most important, and partially buried D29 of NP2 or D30 of NP4 being second in importance. The pKas of the carboxyl groups studied vary significantly, but all are largely deprotonated at pH 7.5 except E124. PMID:19175316

  11. Computational design of a PAK1 binding protein.

    PubMed

    Jha, Ramesh K; Leaver-Fay, Andrew; Yin, Shuangye; Wu, Yibing; Butterfoss, Glenn L; Szyperski, Thomas; Dokholyan, Nikolay V; Kuhlman, Brian

    2010-07-01

    We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 muM. The tightest binding design, named Spider Roll, bound with an affinity of 100 muM. NMR-based structure prediction of Spider Roll based on backbone and (13)C(beta) chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding 'on-rate' constant (<10(5) M(-1) s(-1)). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful

  12. Internal dynamics of DNA - a solid state deuterium NMR study

    SciTech Connect

    Huang, Wen-Chang.

    1989-01-01

    In this dissertation, solid state {sup 2}H NMR spectroscopy has been used to investigate the dynamics of the sodium salt oligonucleotide, (d(CGCGAATTCGCG)){sub 2}, which contains the Eco R1 binding site. Deuterium quadrupole echo line shape and spin-lattice relaxation times were obtained as a function of hydration on three different deuterated samples. In the first sample, (d{sub 12}-(d(CG*CG*A*A*TTCG*CG*)){sub 2}), the C8 proton of all purine in the self-complementary dodecamer were exchanged for deuterons. Specifically labeled thymidine (C6 deuterated) was also synthetically incorporated at the seventh position (counting 5{prime} to 3{prime}) in the sequence (d{sub 2}-(d(CGCGAAT*TCGCG)){sub 2}). In the third sample the C2{double prime} position of the furanose ring of adenosine at the fifth and sixth positions in the same sequence (d{sub 4}-(d(CGCGA*A*TTCGCG)){sub 2}) was deuterium labeled. The static quadrupole coupling constant (e{sup 2}qQ/h) and asymmetry parameter ({eta}) were obtained through the analysis of appropriative motional models from the corresponding monomers studies.

  13. Modern NMR Spectroscopy.

    ERIC Educational Resources Information Center

    Jelinski, Lynn W.

    1984-01-01

    Discusses direct chemical information that can be obtained from modern nuclear magnetic resonance (NMR) methods, concentrating on the types of problems that can be solved. Shows how selected methods provide information about polymers, bipolymers, biochemistry, small organic molecules, inorganic compounds, and compounds oriented in a magnetic…

  14. Some nitrogen-14 NMR studies in solids

    SciTech Connect

    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.

  15. 19F NMR study on the complex of fluorinated vitamin D derivatives with vitamin D receptor: elucidation of the conformation of vitamin D ligands accommodated in the receptor.

    PubMed

    Morizono, Daisuke

    2011-12-28

    Nuclear receptors mediate allosteric communications where ligand binding initiates a cascade of signal transduction. The interaction of vitamin D with vitamin D receptor (VDR) was investigated by (19)F NMR spectroscopy of the complexes of three fluorinated vitamin D derivatives with the full-length rat VDR-LBD. In the (19)F NMR spectra of the VDR-ligand complexes, the A-ring of 4,4-difluoro-1,25(OH)2D3 was revealed to adopt β-conformation in the VDR in solution, and the spectra were shown to be dependent on the dissociation constant. While the complex of 4,4-difluoro-1,25(OH)2D3 with VDR exhibited a clear distinguishable (19)F NMR spectrum, those of (19)F-1,25(OH)2D3 stereoisomers, which have 10-fold higher VDR affinity than 4,4-difluoro-1,25(OH)2D3, did not. The solid-phase NMR technique was useful for (19)F-1,25(OH)2D3 stereoisomers. The fluorinated vitamin D derivatives showed marked changes in the chemical shift (Δ4-19.7 ppm) upon VDR-complex formation, and the ab initio MO method suggested that van der Waals interactions play a major role in the complex formation.

  16. Identification by stopped-exchange solution /sup 31/P NMR spectroscopy of the stepwise formation of (AgL/sub n/)PF/sub 6/ (n = 1-4). Comparison of metal-phosphorus coupling constants for triphenylphosphine and 5-phenyldibenzophosphole

    SciTech Connect

    Alyea, E.C.; Malito, J.; Nelson, J.H.

    1987-12-16

    The coordination properties of 5-phenyldibenzophosphate (PhDBP) have been studied extensively. The stepwise formation of (L/sub n/Ag)/sup +/PF/sub 6//sup -/ (n = 1-4) for L = PhDBP and PhP/sub 3/ observed in situ by stopped-exchange solution /sup 31/P NMR spectroscopy is reported herein. The relative coordinating properties of PhDBP and PhP/sub 3/ are compared. 21 references, 1 figure, 2 tables.

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

    ERIC Educational Resources Information Center

    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…

  18. The anthrax protective antigen (PA63) bound conformation of a peptide inhibitor of the binding of lethal factor to PA63: as determined by trNOESY NMR and molecular modeling.

    PubMed

    Hicks, Rickey P; Bhattacharjee, Apurba K; Koser, Brandon W; Traficante, Daniel D

    2004-10-21

    Anthrax protective antigen (PA) is one of the three proteins produced by the gram positive bacteria Bacillus anthracis collectively known as the "anthrax toxin" (Ascenzi, P.; Visca, P.; Ippolito, G.; Spallarossa, A.; Bolognesi, M.; et al. Anthrax toxin: a tripartite lethal combination. FEBS Lett. 2002, 531, 384-388). The role played by PA in anthrax intoxication is to transport the two enzymes lethal factor (LF) and edema factor (EF) into the cell. Collier and co-workers (Mourez, M.; Kane, R. S.; Mogridge, J.; Metallo, S.; Deschatelets, P.; et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 2001, 958). reported the isolation of two peptides via phage display that bind to the PA63 heptamer and inhibit its interaction with LF and EF, and thereby prevent the transport of LF and EF into the cell. One of these peptides, His-Thr-Ser-Thr-Try-Trp-Trp-Leu-Asp-Gly-Ala-Pro (P1), was selected for structural investigation on the basis of its ability to prevent the binding of LF to the PA63 heptamer bundle. Two-dimensional trNOESY experiments coupled with NOE restrained simulated annealing calculations were used to determine the PA63-bound conformation of P1. On binding to PA63, P1 adopts a helical conformation involving residues 3-9 while the C- and N-terminal residues exhibit dynamic fraying.

  19. NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

    SciTech Connect

    Birdsall, B.; Andrews, J.; Ostler, G.; Tendler, S.J.B.; Feeney, J.; Roberts, G.C.K.; Davies, R.W.; Cheung, H.T.A. )

    1989-02-07

    Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21 {yields} Leu and Asp 26 {yields} Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. {sup 1}H, {sup 13}C, and {sup 31}P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. {sup 1}H NMR studies of the Trp 21 {yields} Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 {angstrom} away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21 {yields} Leu mutant indicate that the delicately poised equilibria can be perturbed. Some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim.

  20. Space Shuttle astrodynamical constants

    NASA Technical Reports Server (NTRS)

    Cockrell, B. F.; Williamson, B.

    1978-01-01

    Basic space shuttle astrodynamic constants are reported for use in mission planning and construction of ground and onboard software input loads. The data included here are provided to facilitate the use of consistent numerical values throughout the project.

  1. The cosmological constant problem

    SciTech Connect

    Dolgov, A.D.

    1989-05-01

    A review of the cosmological term problem is presented. Baby universe model and the compensating field model are discussed. The importance of more accurate data on the Hubble constant and the Universe age is stressed. 18 refs.

  2. Constant potential pulse polarography

    USGS Publications Warehouse

    Christie, J.H.; Jackson, L.L.; Osteryoung, R.A.

    1976-01-01

    The new technique of constant potential pulse polarography, In which all pulses are to be the same potential, is presented theoretically and evaluated experimentally. The response obtained is in the form of a faradaic current wave superimposed on a constant capacitative component. Results obtained with a computer-controlled system exhibit a capillary response current similar to that observed In normal pulse polarography. Calibration curves for Pb obtained using a modified commercial pulse polarographic instrument are in good accord with theoretical predictions.

  3. Binding of carboxylic acids by fluorescent pyridyl ureas.

    PubMed

    Jordan, Lisa M; Boyle, Paul D; Sargent, Andrew L; Allen, William E

    2010-12-17

    Fluorescent pyrid-2-yl ureas were prepared by treating halogenated 2-aminopyridines with hexyl isocyanate, followed by Sonogashira coupling with arylacetylenes. The sensors emit light of ∼360 nm with quantum yields of 0.05-0.1 in acetonitrile solution. Addition of strong organic acids (pK(a) < 13 in CH(3)CN) shifts the fluorescence band to lower energy, and clean isoemissive behavior is observed. Fluorescence response curves (i.e., F/F(0) vs [acid](total)) are hyperbolic in shape for CCl(3)COOH and CF(3)COOH, with association constants on the order of 10(3) M(-1) for both acids. (1)H NMR titrations and DFT analyses indicate that trihaloacetic acids bind in ionized form to the receptors. Pyridine protonation disrupts an intramolecular H-bond, thereby unfolding an array of ureido NH donors for recognition of the corresponding carboxylates. Methanesulfonic acid protonates the sensors, but no evidence for conjugate base binding at the urea moiety is found by NMR. An isosteric control compound that lacks an integrated pyridine does not undergo significant fluorescence changes upon acidification.

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

    SciTech Connect

    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.

  5. Potentiometric and NMR complexation studies of phenylboronic acid PBA and its aminophosphonate analog with selected catecholamines

    NASA Astrophysics Data System (ADS)

    Ptak, Tomasz; Młynarz, Piotr; Dobosz, Agnieszka; Rydzewska, Agata; Prokopowicz, Monika

    2013-05-01

    Boronic acids are a class of intensively explored compounds, which according to their specific properties have been intensively explored in last decades. Among them phenylboronic acids and their derivatives are most frequently examined as receptors for diverse carbohydrates. In turn, there is a large gap in basic research concerning complexation of catecholamines by these compounds. Therefore, we decided to undertake studies on interaction of chosen catecholamines, namely: noradrenaline (norephinephrine), dopamine, L-DOPA, DOPA-P (phosphonic analog of L-DOPA) and catechol, with simple phenyl boronic acid PBA by means of potentiometry and NMR spectroscopy. For comparison, the binding properties of recently synthesized phenylboronic receptor 1 bearing aminophosphonate function in meta-position were investigated and showed promising ability to bind catecholamines. The protonation and stability constants of PBA and receptor 1 complexes were examined by potentiometry. The obtained results demonstrated that PBA binds the catecholamines with the following affinity order: noradrenaline ⩾ dopamine ≈ L-DOPA > catechol > DOPA-P, while its modified analog 1 reveals slightly different preferences: dopamine > noradrenaline > catechol > L-DOPA > DOPA-P.

  6. Predicting Ca2+ -binding sites using refined carbon clusters.

    PubMed

    Zhao, Kun; Wang, Xue; Wong, Hing C; Wohlhueter, Robert; Kirberger, Michael P; Chen, Guantao; Yang, Jenny J

    2012-12-01

    Identifying Ca(2+) -binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca(2+) -binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca(2+) -binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca(2+) -binding site. Similarly, both Ca(2+) and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca(2+) -binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUG(C) ) to predict Ca(2+) -binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand co-ordinates, MUG(C) is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures composed of 43 Ca(2+) -binding proteins. Additionally, prediction of Ca(2+) -binding sites in NMR structures was obtained by MUG(C) using a different set of parameters, which were determined by the analysis of both Ca(2+) -constrained and unconstrained Ca(2+) -loaded structures derived from NMR data. MUG(C) identified 20 of 21 Ca(2+) -binding sites in NMR structures inferred without the use of Ca(2+) constraints. MUG(C) predictions are also highly selective for Ca(2+) -binding sites as analyses of binding sites for Mg(2+) , Zn(2+) , and Pb(2+) were not identified as Ca(2+) -binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient not only for accurate identification of Ca(2+) -binding sites in NMR and X-ray structures but also for selective differentiation between Ca(2+) and other relevant divalent cations.

  7. Structural basis of the tight binding of pyridoxal 5'-phosphate to a low molecular weight protein tyrosine phosphatase.

    PubMed

    Zhou, M; Van Etten, R L

    1999-03-01

    Pyridoxal 5'-phosphate (PLP) binds tightly to bovine low Mr protein tyrosine phosphatase (BPTP), but it is a very poor substrate for the enzyme. The structural basis of this tight binding of PLP is examined here by a variety of methods. Binding constants of a number of PLP analogues were measured with wild-type BPTP, and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The tight binding of PLP (Ki = 7.6 microM) causes a downfield shift of the His-72 Cepsilon1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR spectroscopy with an MLEV-17 spectral editing scheme was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. UV-visible spectrophotometric titration also supported this conclusion. The binding constant of PLP to a C17A mutant was similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is "essential" for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. It is concluded that Asp-129 binds to the pyridinium nitrogen of PLP and that this renders Asp-129 effectively unavailable to serve its essential catalytic role as a general acid. The interactions described here should be useful in the design of specific

  8. Live cell NMR.

    PubMed

    Freedberg, Darón I; Selenko, Philipp

    2014-01-01

    Ever since scientists realized that cells are the basic building blocks of all life, they have been developing tools to look inside them to reveal the architectures and mechanisms that define their biological functions. Whereas "looking into cells" is typically said in reference to optical microscopy, high-resolution in-cell and on-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful method that offers exciting new possibilities for structural and functional studies in and on live cells. In contrast to conventional imaging techniques, in- and on-cell NMR methods do not provide spatial information on cellular biomolecules. Instead, they enable atomic-resolution insights into the native cell states of proteins, nucleic acids, glycans, and lipids. Here we review recent advances and developments in both fields and discuss emerging concepts that have been delineated with these methods.

  9. The NMR phased array.

    PubMed

    Roemer, P B; Edelstein, W A; Hayes, C E; Souza, S P; Mueller, O M

    1990-11-01

    We describe methods for simultaneously acquiring and subsequently combining data from a multitude of closely positioned NMR receiving coils. The approach is conceptually similar to phased array radar and ultrasound and hence we call our techniques the "NMR phased array." The NMR phased array offers the signal-to-noise ratio (SNR) and resolution of a small surface coil over fields-of-view (FOV) normally associated with body imaging with no increase in imaging time. The NMR phased array can be applied to both imaging and spectroscopy for all pulse sequences. The problematic interactions among nearby surface coils is eliminated (a) by overlapping adjacent coils to give zero mutual inductance, hence zero interaction, and (b) by attaching low input impedance preamplifiers to all coils, thus eliminating interference among next nearest and more distant neighbors. We derive an algorithm for combining the data from the phased array elements to yield an image with optimum SNR. Other techniques which are easier to implement at the cost of lower SNR are explored. Phased array imaging is demonstrated with high resolution (512 x 512, 48-cm FOV, and 32-cm FOV) spin-echo images of the thoracic and lumbar spine. Data were acquired from four-element linear spine arrays, the first made of 12-cm square coils and the second made of 8-cm square coils. When compared with images from a single 15 x 30-cm rectangular coil and identical imaging parameters, the phased array yields a 2X and 3X higher SNR at the depth of the spine (approximately 7 cm). PMID:2266841

  10. NMR imaging of materials

    SciTech Connect

    Vinegar, H.J.; Rothwell, W.P.

    1988-03-01

    A method for obtaining at least one petrophysical property of a porous material containing therein at least one preselected fluid, is described, comprising: NMR imaging the material to generate signals dependent upon both M(0) and T/sub 1/ and M(0) and T/sub 2/, generating separate M(0), T/sub 1/ and T/sub 2/ images from the signals, and determining at least one petrophysical property from at least one of the images.

  11. Determination of the solution-bound conformation of an amino acid binding protein by NMR paramagnetic relaxation enhancement: use of a single flexible paramagnetic probe with improved estimation of its sampling space.

    PubMed

    Bermejo, Guillermo A; Strub, Marie-Paule; Ho, Chien; Tjandra, Nico

    2009-07-15

    We demonstrate the feasibility of elucidating the bound ("closed") conformation of a periplasmic binding protein, the glutamine-binding protein (GlnBP), in solution, using paramagnetic relaxation enhancements (PREs) arising from a single paramagnetic group. GlnBP consists of two globular domains connected by a hinge. Using the ligand-free ("open") conformation as a starting point, conjoined rigid-body/torsion-angle simulated annealing calculations were performed using backbone (1)H(N)-PREs as a major source of distance information. Paramagnetic probe flexibility was accounted for via a multiple-conformer representation. A conventional approach where the entire PRE data set is enforced at once during simulated annealing yielded poor results due to inappropriate conformational sampling of the probe. On the other hand, significant improvements in coordinate accuracy were obtained by estimating the probe sampling space prior to structure calculation. Such sampling is achieved by refining the ensemble of probe conformers with intradomain PREs only, keeping the protein backbone fixed in the open form. Subsequently, while constraining the probe to the previously found conformations, the domains are allowed to move relative to each other under the influence of the non-intradomain PREs, giving the hinge region torsional degrees of freedom. Thus, by partitioning the protocol into "probe sampling" and "backbone sampling" stages, structures significantly closer to the X-ray structure of ligand-bound GlnBP were obtained.

  12. Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

    PubMed Central

    Hammann, Christian; Norman, David G.; Lilley, David M. J.

    2001-01-01

    We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range. PMID:11331743

  13. Variation of Fundamental Constants

    NASA Astrophysics Data System (ADS)

    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.

  14. Characterization of lithium coordination sites with magic-angle spinning NMR

    NASA Astrophysics Data System (ADS)

    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

  15. Characterization of lithium coordination sites with magic-angle spinning NMR.

    PubMed

    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

  16. Sodium ion effect on silk fibroin conformation characterized by solid-state NMR and generalized 2D NMR NMR correlation

    NASA Astrophysics Data System (ADS)

    Ruan, Qing-Xia; Zhou, Ping

    2008-07-01

    In the present work, we investigated Na + ion effect on the silk fibroin (SF) conformation. Samples are Na +-involved regenerated silk fibroin films. 13C CP-MAS NMR demonstrates that as added [Na +] increases, partial silk fibroin conformation transit from helix-form to β-form at certain Na + ion concentration which is much higher than that in Bombyx mori silkworm gland. The generalized two-dimensional NMR-NMR correlation analysis reveals that silk fibroin undergoes several intermediate states during its conformation transition process as [Na +] increase. The appearance order of the intermediates is followed as: helix and/or random coil → helix-like → β-sheet-like → β-sheet, which is the same as that produced by pH decrease from 6.8 to 4.8 in the resultant regenerated silk fibroin films. The binding sites of Na + to silk fibroin might involve the carbonyl oxygen atom of certain amino acids sequence which could promote the formation of β-sheet conformation. Since the Na +sbnd O bond is weak, the ability of Na + inducing the secondary structure transition is weaker than those of Ca 2+, Cu 2+ and even K +. It is maybe a reason why the sodium content is much lower than potassium in the silkworm gland.

  17. Elastic constants of calcite

    USGS Publications Warehouse

    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.

  18. Soils, Pores, and NMR

    NASA Astrophysics Data System (ADS)

    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 < 5 ms. Moreover, the dependence of the relaxation rate on magnetic field strength allows the identification of 2D diffusion at the interfaces as the mechanism which governs the relaxation process (Pohlmeier et al. 2009). T2 relaxation curves are frequently measured for the rapid characterization of soils by means of the CPMG echo train. Basically, they contain the same information about the pore systems like T1 curves, since mostly the overall relaxation is dominated by surface relaxivity and the surface/volume ratio of the pores. However, one must be aware that T2 relaxation is additionally affected by diffusion in internal gradients, and this can be overcome by using sufficiently short echo times and low magnetic fields (Stingaciu et al. 2009). Second, the logic continuation of conventional relaxation measurements is the 2-dimensional experiment, where prior to the final detection of the CPMG echo train an encoding period is applied. This can be T1-encoding by an inversion pulse, or T2 encoding by a sequence of 90 and 180° pulses. During the following evolution time the separately encoded signals can mix and this reveals information about

  19. The Hubble constant

    NASA Technical Reports Server (NTRS)

    Huchra, John P.

    1992-01-01

    The Hubble constant is the constant of proportionality between recession velocity and distance in the expanding universe. It is a fundamental property of cosmology that sets both the scale and the expansion age of the universe. It is determined by measurement of galaxy radial velocities and distances. Although there has been considerable progress in the development of new techniques for the measurements of galaxy distances, both calibration uncertainties and debates over systematic errors remain. Current determinations still range over nearly a factor of 2; the higher values favored by most local measurements are not consistent with many theories of the origin of large-scale structure and stellar evolution.

  20. Bayesian reconstruction of projection reconstruction NMR (PR-NMR).

    PubMed

    Yoon, Ji Won

    2014-11-01

    Projection reconstruction nuclear magnetic resonance (PR-NMR) is a technique for generating multidimensional NMR spectra. A small number of projections from lower-dimensional NMR spectra are used to reconstruct the multidimensional NMR spectra. In our previous work, it was shown that multidimensional NMR spectra are efficiently reconstructed using peak-by-peak based reversible jump Markov chain Monte Carlo (RJMCMC) algorithm. We propose an extended and generalized RJMCMC algorithm replacing a simple linear model with a linear mixed model to reconstruct close NMR spectra into true spectra. This statistical method generates samples in a Bayesian scheme. Our proposed algorithm is tested on a set of six projections derived from the three-dimensional 700 MHz HNCO spectrum of a protein HasA. PMID:25218584

  1. Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

    PubMed

    Norne, J E; Hjalmarsson, S G; Lindman, B; Zeppezauer, M

    1975-07-29

    The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more

  2. Compassion is a constant.

    PubMed

    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.

  3. XrayOpticsConstants

    2005-06-20

    This application (XrayOpticsConstants) is a tool for displaying X-ray and Optical properties for a given material, x-ray photon energy, and in the case of a gas, pressure. The display includes fields such as the photo-electric absorption attenuation length, density, material composition, index of refraction, and emission properties (for scintillator materials).

  4. Compassion is a constant.

    PubMed

    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. PMID:26542898

  5. Metal binding to the HIV nucleocapsid peptide.

    PubMed

    McLendon, G; Hull, H; Larkin, K; Chang, W

    1999-04-01

    Co(II) and Zn(II) binding constants have been measured for binding to the HIV-1 nucleocapsid N-terminal metal binding domain (residues 1-18), using competition titration methods and monitoring Co(II) binding by visible absorbance spectroscopy. Enthalpies for binding were directly measured by isothermal titration colorimetry. The results are compared with recent studies of related systems, including a study of Zn(II) binding by the full length protein.

  6. p53 and TFIIEα share a common binding site on the Tfb1/p62 subunit of TFIIH

    PubMed Central

    Di Lello, Paola; Miller Jenkins, Lisa M.; Mas, Caroline; Langlois, Chantal; Malitskaya, Elena; Fradet-Turcotte, Amélie; Archambault, Jacques; Legault, Pascale; Omichinski, James G.

    2008-01-01

    The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the α-subunit of the general transcription factor IIE (TFIIEα). We have determined that the acidic carboxyl terminus of TFIIEα (TFIIEα336–439) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEα binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEα336–439 is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEα336–439 and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEα336–439 contains a small domain (residues 395–433) folded in a novel ββααα topology. NMR mapping studies demonstrate that two unstructured regions (residues 377–393 and residues 433–439) located on either side of the folded domain appear to be required for TFIIEα336–439 binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEα336–439 can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEα and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53. PMID:18160537

  7. p53 and TFIIEalpha share a common binding site on the Tfb1/p62 subunit of TFIIH.

    PubMed

    Di Lello, Paola; Miller Jenkins, Lisa M; Mas, Caroline; Langlois, Chantal; Malitskaya, Elena; Fradet-Turcotte, Amélie; Archambault, Jacques; Legault, Pascale; Omichinski, James G

    2008-01-01

    The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53. PMID:18160537

  8. Conformational analysis of small molecules: NMR and quantum mechanics calculations.

    PubMed

    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.

  9. Conformational analysis of small molecules: NMR and quantum mechanics calculations.

    PubMed

    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. PMID:27573182

  10. Multinuclear NMR studies of relaxor ferroelectrics

    NASA Astrophysics Data System (ADS)

    Zhou, Donghua

    Multinuclear NMR of 93Nb, 45Sc, and 207Pb has been carried out to study the structure, disorder, and dynamics of a series of important solid solutions: perovskite relaxor ferroelectric materials (1-x) Pb(Mg1/3Nb 2/3)O3-x Pb(Sc1/2Nb1/2)O 3 (PMN-PSN). 93Nb NMR investigations of the local structure and cation order/disorder are presented as a function of PSN concentration, x. The superb fidelity and accuracy of 3QMAS allows us to make clear and consistent assignments of spectral intensities to the 28 possible nearest B-site neighbor (nBn) configurations, (NMg, NSc, NNb), where each number ranges from 0 to 6 and their sum is 6. For most of the 28 possible nBn configurations, isotropic chemical shifts and quadrupole product constants have been extracted from the data. The seven configurations with only larger cations, Mg 2+ and Sc3+ (and no Nb5+) are assigned to the seven observed narrow peaks, whose deconvoluted intensities facilitate quantitative evaluation of, and differentiation between, different models of B-site (chemical) disorder. The "completely random" model is ruled out and the "random site" model is shown to be in qualitative agreement with the NMR experiments. To obtain quantitative agreement with observed NMR intensities, the random site model is slightly modified by including unlike-pair interaction energies. To date, 45Sc studies have not been as fruitful as 93Nb NMR because the resolution is lower in the 45Sc spectra. The lower resolution of 45Sc spectra is due to a smaller span of isotropic chemical shift (40 ppm for 45Sc vs. 82 ppm for 93Nb) and to the lack of a fortuitous mechanism that simplifies the 93Nb spectra; for 93Nb the overlap of the isotropic chemical shifts of 6-Sc and 6-Nb configurations results in the alignment of all the 28 configurations along only seven quadrupole distribution axes. Finally we present variable temperature 207Pb static, MAS, and 2D-PASS NMR studies. Strong linear correlations between isotropic and anisotropic chemical

  11. NMR Studies on Li+, Na+ and K+ Complexes of Orthoester Cryptand o-Me2-1.1.1

    PubMed Central

    Brachvogel, René-Chris; Maid, Harald; von Delius, Max

    2015-01-01

    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. PMID:26334274

  12. High-Resolution NMR Studies of Human Tissue Factor

    PubMed Central

    Nuzzio, Kristin M.; Watt, Eric D.; Boettcher, John M.; Gajsiewicz, Joshua M.; Morrissey, James H.; Rienstra, Chad M.

    2016-01-01

    In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation. PMID:27657719

  13. Conjoined Use of EM and NMR in RNA Structure Refinement

    PubMed Central

    Gong, Zhou; Schwieters, Charles D.; Tang, Chun

    2015-01-01

    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 (ΨCD)2 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. PMID:25798848

  14. 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.

    NASA Astrophysics Data System (ADS)

    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.

  15. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  16. Some specific features of the NMR study of fluid flows

    NASA Astrophysics Data System (ADS)

    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.

  17. NMR Tube Degradation Method for Sugar Analysis of Glycosides.

    PubMed

    Giner, José-Luis; Feng, Ju; Kiemle, David J

    2016-09-23

    The sugar subunits of natural glycosides can be conveniently determined by acid hydrolysis and (1)H NMR spectroscopy without isolation or derivatization. The chemical shifts, coupling constants, and integral ratios of the anomeric signals allow each monosaccharide to be identified and its molar ratio to other monosaccharides to be quantified. The NMR data for the anomeric signals of 28 monosaccharides and three disaccharides are reported. Application of the method is demonstrated with the flavonoid glycoside naringin (1), the aminoglycoside antibiotics kanamycin (2) and tobramycin (3), and the saponin digitonin (4). PMID:27603739

  18. Wall of fundamental constants

    SciTech Connect

    Olive, Keith A.; Peloso, Marco; Uzan, Jean-Philippe

    2011-02-15

    We consider the signatures of a domain wall produced in the spontaneous symmetry breaking involving a dilatonlike scalar field coupled to electromagnetism. Domains on either side of the wall exhibit slight differences in their respective values of the fine-structure constant, {alpha}. If such a wall is present within our Hubble volume, absorption spectra at large redshifts may or may not provide a variation in {alpha} relative to the terrestrial value, depending on our relative position with respect to the wall. This wall could resolve the contradiction between claims of a variation of {alpha} based on Keck/Hires data and of the constancy of {alpha} based on Very Large Telescope data. We derive the properties of the wall and the parameters of the underlying microscopic model required to reproduce the possible spatial variation of {alpha}. We discuss the constraints on the existence of the low-energy domain wall and describe its observational implications concerning the variation of the fundamental constants.

  19. Varying constants quantum cosmology

    SciTech Connect

    Leszczyńska, Katarzyna; Balcerzak, Adam; Dabrowski, Mariusz P. E-mail: abalcerz@wmf.univ.szczecin.pl

    2015-02-01

    We discuss minisuperspace models within the framework of varying physical constants theories including Λ-term. In particular, we consider the varying speed of light (VSL) theory and varying gravitational constant theory (VG) using the specific ansätze for the variability of constants: c(a) = c{sub 0} a{sup n} and G(a)=G{sub 0} a{sup q}. We find that most of the varying c and G minisuperspace potentials are of the tunneling type which allows to use WKB approximation of quantum mechanics. Using this method we show that the probability of tunneling of the universe ''from nothing'' (a=0) to a Friedmann geometry with the scale factor a{sub t} is large for growing c models and is strongly suppressed for diminishing c models. As for G varying, the probability of tunneling is large for G diminishing, while it is small for G increasing. In general, both varying c and G change the probability of tunneling in comparison to the standard matter content (cosmological term, dust, radiation) universe models.

  20. 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.

    PubMed

    Yan, H G; Tsai, M D

    1991-06-01

    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)

  1. β-cyclodextrin and caffeine complexes with natural polyphenols from olive and olive oils: NMR, thermodynamic, and molecular modeling studies.

    PubMed

    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.

  2. 1H NMR spectroscopic characterization of inclusion complexes of tolfenamic and flufenamic acids with β-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Floare, C. G.; Pirnau, A.; Bogdan, M.

    2013-07-01

    The complexation between the anionic forms of tolfenamic acid and flufenamic acid with β-cyclodextrin was investigated in solution by 1D and 2D proton NMR spectroscopy. The stoichiometry of the complexes was determined by the method of continuous variation using the chemical induced shifts of both the host and guest protons. An analysis of the spectroscopic data revealed that simultaneous inclusion of both rings of tolfenamic and flufenamic acids occur, giving rise each to two isomeric 1:1 complexes. The view of a bimodal binding between these two drugs and β-cyclodextrin was also supported by ROESY experiments. Using a rough approximation, we have estimated the association constants order of magnitude of the 1:1 complexes.

  3. Binding of the growth factor glycyl-L-histidyl-L-lysine by heparin.

    PubMed

    Rabenstein, D L; Robert, J M; Hari, S

    1995-12-01

    Evidence is presented that the growth factor glycyl-histidyl-lysine (GHK) binds to heparin, and the interaction has been characterized by [1H]NMR spectroscopy. 1H chemical shifts indicate that GHK interacts with both the carboxylic acid and the carboxylate forms of heparin. The chemical shift data are consistent with a weak delocalized binding of the triprotonated (ImH+, GlyNH3+, LysNH3+) form of GHK by the carboxylic acid form of heparin. As the pD is increased and the carboxylic acid groups are titrated, chemical shift data indicate that ammonium groups of GHK are hydrogen bonded to heparin carboxylate groups, while the histidyl imidazolium ring occupies the imidazolium-binding site of heparin. Evidence for site-specific binding includes displacement of chemical shift titration curves for heparin to lower pD, increased shielding of specific heparin protons by the imidazolium ring current and displacement of chemical shift titration curves for GHK to higher pD. Specific binding constants were determined for binding of the (ImH+, GlyNH3+), LysNH3+) forms of GHK by the carboxylate form of heparin from chemical shift vs. pD titration data. PMID:7498545

  4. Interaction Between Drugs and Biomedical Materials i: Binding Position of Bezafibrate to Human Serum Alubmin

    NASA Astrophysics Data System (ADS)

    Tanaka, Masami; Minagawa, Keiji; Berber, Mohamed R.; Hafez, Inas H.; Mori, Takeshi

    The interaction between bezafibrate (BZF) and human serum albumin (HSA) was investigated by equilibrium dialysis. Since the binding constant of BZF to HSA was independent of ionic strength and decreased with the addition of fatty acid, the interaction between BZF and HSA was considered to be due to hydrophobic mechanism. Chemical shifts in 1H-NMR spectra of BZF were independent of the concentration of BZF and addition of HSA. Spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) of respective protons of BZF were independent of the concentration, but depended on the concentration of HSA added. The binding position of BZF to HSA was considered to involve the hydrophobic aromatic moiety of BZF from the ratio of spin-spin relaxation rates (1/T2) of BZF bound to HSA and free BZF.

  5. Achievement of 1020MHz NMR.

    PubMed

    Hashi, Kenjiro; Ohki, Shinobu; Matsumoto, Shinji; Nishijima, Gen; Goto, Atsushi; Deguchi, Kenzo; Yamada, Kazuhiko; Noguchi, Takashi; Sakai, Shuji; Takahashi, Masato; Yanagisawa, Yoshinori; Iguchi, Seiya; Yamazaki, Toshio; Maeda, Hideaki; Tanaka, Ryoji; Nemoto, Takahiro; Suematsu, Hiroto; Miki, Takashi; Saito, Kazuyoshi; Shimizu, Tadashi

    2015-07-01

    We have successfully developed a 1020MHz (24.0T) NMR magnet, establishing the world's highest magnetic field in high resolution NMR superconducting magnets. The magnet is a series connection of LTS (low-Tc superconductors NbTi and Nb3Sn) outer coils and an HTS (high-Tc superconductor, Bi-2223) innermost coil, being operated at superfluid liquid helium temperature such as around 1.8K and in a driven-mode by an external DC power supply. The drift of the magnetic field was initially ±0.8ppm/10h without the (2)H lock operation; it was then stabilized to be less than 1ppb/10h by using an NMR internal lock operation. The full-width at half maximum of a (1)H spectrum taken for 1% CHCl3 in acetone-d6 was as low as 0.7Hz (0.7ppb), which was sufficient for solution NMR. On the contrary, the temporal field stability under the external lock operation for solid-state NMR was 170ppb/10h, sufficient for NMR measurements for quadrupolar nuclei such as (17)O; a (17)O NMR measurement for labeled tri-peptide clearly demonstrated the effect of high magnetic field on solid-state NMR spectra. PMID:25978708

  6. THz Dynamic Nuclear Polarization NMR

    PubMed Central

    Nanni, Emilio A.; Barnes, Alexander B.; Griffin, Robert G.; Temkin, Richard J.

    2013-01-01

    Dynamic nuclear polarization (DNP) increases the sensitivity of nuclear magnetic resonance (NMR) spectroscopy by using high frequency microwaves to transfer the polarization of the electrons to the nuclear spins. The enhancement in NMR sensitivity can amount to a factor of well above 100, enabling faster data acquisition and greatly improved NMR measurements. With the increasing magnetic fields (up to 23 T) used in NMR research, the required frequency for DNP falls into the THz band (140–600 GHz). Gyrotrons have been developed to meet the demanding specifications for DNP NMR, including power levels of tens of watts; frequency stability of a few megahertz; and power stability of 1% over runs that last for several days to weeks. Continuous gyrotron frequency tuning of over 1 GHz has also been demonstrated. The complete DNP NMR system must include a low loss transmission line; an optimized antenna; and a holder for efficient coupling of the THz radiation to the sample. This paper describes the DNP NMR process and illustrates the THz systems needed for this demanding spectroscopic application. THz DNP NMR is a rapidly developing, exciting area of THz science and technology. PMID:24639915

  7. Change is a Constant.

    PubMed

    Lubowitz, James H; Provencher, Matthew T; Brand, Jefferson C; Rossi, Michael J; Poehling, Gary G

    2015-06-01

    In 2015, Henry P. Hackett, Managing Editor, Arthroscopy, retires, and Edward A. Goss, Executive Director, Arthroscopy Association of North America (AANA), retires. Association is a positive constant, in a time of change. With change comes a need for continuing education, research, and sharing of ideas. While the quality of education at AANA and ISAKOS is superior and most relevant, the unique reason to travel and meet is the opportunity to interact with innovative colleagues. Personal interaction best stimulates new ideas to improve patient care, research, and teaching. Through our network, we best create innovation.

  8. Cosmology with varying constants.

    PubMed

    Martins, Carlos J A P

    2002-12-15

    The idea of possible time or space variations of the 'fundamental' constants of nature, although not new, is only now beginning to be actively considered by large numbers of researchers in the particle physics, cosmology and astrophysics communities. This revival is mostly due to the claims of possible detection of such variations, in various different contexts and by several groups. I present the current theoretical motivations and expectations for such variations, review the current observational status and discuss the impact of a possible confirmation of these results in our views of cosmology and physics as a whole.

  9. Squaring cooperative binding circles

    PubMed Central

    Deutman, Alexander B. C.; Monnereau, Cyrille; Moalin, Mohamed; Coumans, Ruud G. E.; Veling, Nico; Coenen, Michiel; Smits, Jan M. M.; de Gelder, René; Elemans, Johannes A. A. W.; Ercolani, Gianfranco; Nolte, Roeland J. M.; Rowan, Alan E.

    2009-01-01

    The cooperative binding effects of viologens and pyridines to a synthetic bivalent porphyrin receptor are used as a model system to study how the magnitudes of these effects relate to the experimentally obtained values. The full thermodynamic and kinetic circles concerning both activation and inhibition of the cage of the receptor for the binding of viologens were measured and evaluated. The results strongly emphasize the apparent character of measured binding and rate constants, in which the fractional saturation of receptors with other guests is linearly expressed in these constants. The presented method can be used as a simple tool to better analyze and comprehend the experimentally observed kinetics and thermodynamics of natural and artificial cooperative systems. PMID:19470643

  10. In-cell NMR in E. coli to Monitor Maturation Steps of hSOD1

    PubMed Central

    Banci, Lucia; Barbieri, Letizia; Bertini, Ivano; Cantini, Francesca; Luchinat, Enrico

    2011-01-01

    In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes. PMID:21887272

  11. Study of the New Pulse NMR System for the Jefferson Lab Helium-3 Polarized Target

    NASA Astrophysics Data System (ADS)

    Newton, Joseph

    2013-10-01

    At Jefferson Lab, a polarized Helium-3 target is used to study the neutron. The Helium-3 target is undergoing an upgrade to improve its polarization. Measuring it involved a new technique known as pulse Nuclear Magnetic Resonance (NMR). The focus of this project was to find noise in the Pulse NMR signal and to compute the calibration constant to make the polarization easier to deduce. Pulse NMR calibration tests were performed by doing AFP NMR measurements followed by Pulse NMR measurements while varying certain conditions. These included the convection heater, the operation of the oven, and the operation of the laser. Data analysis was done by fitting the pulse NMR signal from the oscilloscope and utilizing the Fourier Transform. Noise was analyzed in the fitting and the Fourier Transform. The calibration constants were affected by the convection heater. The values deviated between the pumping and target chambers of the cell when there was no convection but the values were closer when convection was induced. As far as the noise, it was found to be significant. These results will enable the calculation of the polarization with pulse NMR. In addition, the signal analysis provided insight into the influence of background noise on the pulse NMR measurement. This research was done though the SULI program of the Department of Energy.

  12. Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody

    SciTech Connect

    Glaudemans, C.P.J.; Lerner, L.; Daves, G.D. Jr.; Kovac, P.; Bax, A. ); Venable, R. )

    1990-12-01

    Transferred nulcear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-{beta}-D-galactopyranosyl(1{yields}6)-4-deoxy-4-fluoro-{beta}-D galactopyranoside (me4FGal{sub 2}) and its selectively deuteriated analogue, methyl O-{beta}-D-galactopyranosyl(1{yields}6)-4-deoxy-2-deuterio-4-fluoro-{beta}-D-galactopyranoside (me4F2dGal{sub 2}). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal{sub 2} was inferred from measurements of vicinal {sup 1}H-{sup 1}H coupling constants, long-range {sup 1}H-{sup 13}C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of {sup 1}H-{sup 1}H and {sup 1}H-{sup 13}C constants indicates that the major conformer has an extended conformation. TRNOE measurements on me4FGal{sub 2} and me4F2dGal{sub 2} in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints. A new method, based on changes in the fluorine longitudinal relaxation rate, is used to measure the ligand-antibody dissociation rate constant.

  13. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed

    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.

  14. NMR investigation of the complexation and chiral discrimination of pyrazole sulfonamide derivatives with cyclodextrins.

    PubMed

    Rogez-Florent, Tiphaine; Azaroual, Nathalie; Goossens, Laurence; Goossens, Jean-François; Danel, Cécile

    2015-01-22

    The complexes formed between six original chiral diaryl-pyrazole sulfonamide derivatives, displaying poor solubility, and various CDs (native α-, β- and γ-CDs, hydroxypropylated HP-β-CD, methylated Me-β-CD or amino NH2-β-CD) were studied by 1D and 2D (1)H NMR at physiological pH in order to determine their apparent binding constant, stoichiometry and structure of the supramolecular assembly. For some complexes, the spectra obtained for free racemic compound and for racemic compound in presence of CD indicate a splitting of signal(s). Additional experiments with pure enantiomer and enriched enantiomer allow us to attribute this behavior to chiral discrimination. The complexing ability of the native β-CD towards our compounds appears the most promising since binding values around 7×10(2)M(-1) are obtained. The two-dimensional ROESY ((1)H-(1)H) experiments prove the inclusion of the aliphatic part of the compound in the CD cavity. It is noteworthy that this inclusion occurs via the smaller opening of the cavity.

  15. The Determination of Protonation Constants of Peptidomimetic Cyclophanes in Binary Methanol-Water Mixtures

    PubMed Central

    Tomczyk, Danuta; Andrijewski, Grzegorz

    2016-01-01

    The protonation constants of new group of peptidomimetic cyclophanes with valine or phenylalanine moieties incorporated into the macrocyclic skeleton as well as their linear analogues were determined by potentiometric measurements in solutions of methanol-water mixtures at 25°C and constant ionic strength. The influence of cavity size, location of protonation sites, and attached substituents of the macrocyclic ligands on the protonation constants were discussed on the basis of potentiometric measurement as well as H1-NMR results. PMID:27516918

  16. The Determination of Protonation Constants of Peptidomimetic Cyclophanes in Binary Methanol-Water Mixtures.

    PubMed

    Seliger, Piotr; Tomczyk, Danuta; Andrijewski, Grzegorz; Tomal, Ewa

    2016-01-01

    The protonation constants of new group of peptidomimetic cyclophanes with valine or phenylalanine moieties incorporated into the macrocyclic skeleton as well as their linear analogues were determined by potentiometric measurements in solutions of methanol-water mixtures at 25°C and constant ionic strength. The influence of cavity size, location of protonation sites, and attached substituents of the macrocyclic ligands on the protonation constants were discussed on the basis of potentiometric measurement as well as H(1)-NMR results. PMID:27516918

  17. Developing SABRE as an analytical tool in NMR

    NASA Astrophysics Data System (ADS)

    Lloyd, Lyrelle Stacey

    Work presented in this thesis centres around the application of the new hyperpolarisation technique, SABRE, within nuclear magnetic resonance spectroscopy, focusing on optimisation of the technique to characterise small organic molecules. While pyridine was employed as a model substrate, studies on a range of molecules are investigated including substituted pyridines, quinolines, thiazoles and indoles are detailed. Initial investigations explored how the properties of the SABRE catalyst effect the extent of polarisation transfer exhibited. The most important of these properties proved to be the rate constants for loss of pyridine and hydrides as these define the contact time of pyridine with the parahydrogen derived hydride ligands in the metal template. The effect of changing the temperature, solvent or concentration of substrate or catalyst are rationalised. For instance, the catalyst ICy(a) exhibits relatively slow ligand exchange rates and increasing the temperature during hyperpolarisation increases the observed signal enhancements. These studies have revealed a second polarisation transfer template can be used with SABRE in which two substrate molecules are bound. This allows the possibility of investigation of larger substrates which might otherwise be too sterically encumbered to bind. Another significant advance relates to the first demonstration that SABRE can be used in conjunction with an automated system designed with Bruker allowing the acquisition of scan averaged, phase cycled and traditional 2D spectra. The system also allowed investigations into the effect of the polarisation transfer field and application of that knowledge to collect single-scan 13C data for characterisation. The successful acquisition of 1H NOESY, 1H-1H COSY, 1H-13C 2D and ultrafast 1H-1H COSY NMR sequences is detailed for a 10 mM concentration sample, with 1H data collected for a 1 mM sample. A range of studies which aim to demonstrate the applicability of SABRE to the

  18. Vinculin Tail Dimerization and Paxillin Binding

    NASA Astrophysics Data System (ADS)

    Campbell, Sharon

    2006-03-01

    Vinculin is a highly conserved cytoskeletal protein that is essential for regulation of cell morphology and migration, and is a critical component of both cell-cell and cell-matrix complexes. The tail domain of vinculin (Vt) was crystallized as a homodimer and is believed to bind F-actin as a dimer. We have characterized Vt dimerization by Nuclear Magnetic Resonance (NMR) Spectroscopy and identified the dimer interface in solution by chemical shift perturbation. The Vt dimer interface in solution is similar to the crystallographic dimer interface. Interestingly, the Vt dimer interface determined by NMR partially overlaps the paxillin binding region previously defined coarsely by deletion mutagenesis and gel-blot assays. To further characterize the paxillin binding site in Vt and probe relationship between paxillin binding and dimerization, we conducted chemical shift perturbations experiments using a paxillin derived peptide, LD2. Our NMR experiments have confirmed that the paxillin binding site and the Vt dimerization site partially overlap, and we have further characterized both of these two binding interfaces. Information derived from these studies was used to identify mutations in Vt that selectively perturb paxillin binding and Vt self-association. These mutants are currently being characterized for their utility in structural and biological analyses to elucidate the role of paxillin binding and Vt dimerization in vinculin function.

  19. About variable constants

    NASA Astrophysics Data System (ADS)

    Blichert-Toft, J.; Albarede, F.

    2011-12-01

    When only modern isotope compositions are concerned, the choice of normalization values is inconsequential provided that their values are universally accepted. No harm is done as long as large amounts of standard reference material with known isotopic differences with respect to the reference value ('anchor point') can be maintained under controlled conditions. For over five decades, the scientific community has been referring to an essentially unavailable SMOW for stable O and H isotopes and to a long-gone belemnite sample for carbon. For radiogenic isotopes, the isotope composition of the daughter element, the parent-daughter ratio, and a particular value of the decay constant are all part of the reference. For the Lu-Hf system, for which the physical measurements of the decay constant have been particularly defective, the reference includes the isotope composition of Hf and the Lu/Hf ratio of an unfortunately heterogeneous chondrite mix that has been successively refined by Patchett and Tatsumoto (1981), Blichert-Toft and Albarede (1997, BTA), and Bouvier et al. (2008, BVP). The \\varepsilonHf(T) difference created by using BTA and BVP is nearly within error (+0.45 epsilon units today and -0.36 at 3 Ga) and therefore of little or no consequence. A more serious issue arises when the chondritic reference is taken to represent the Hf isotope evolution of the Bulk Silicate Earth (BSE): the initial isotope composition of the Solar System, as determined by the indistinguishable intercepts of the external eucrite isochron (Blichert-Toft et al., 2002) and the internal angrite SAH99555 isochron (Thrane et al., 2010), differs from the chondrite value of BTA and BVP extrapolated to 4.56 Ga by ~5 epsilon units. This difference and the overestimated value of the 176Lu decay constant derived from the slopes of these isochrons, have been interpreted as reflecting irradiation of the solar nebula by either gamma (Albarede et al., 2006) or cosmic rays (Thrane et al., 2010) during

  20. A tricatecholic receptor for carbohydrate recognition: synthesis and binding studies.

    PubMed

    Cacciarini, Martina; Cordiano, Elisa; Nativi, Cristina; Roelens, Stefano

    2007-05-11

    A new tripodal receptor bearing three catechol subunits on a benzene platform has been synthesized in four steps from 1,3,5-triethylbenzene and pyrogallol. The binding ability of the tricatecholic receptor was investigated toward several monosaccharides in CDCl3, where multiple equilibria were detected, and compared to that of a previously reported trisureidic receptor of analogous structure. Association constants were measured by 1H NMR titrations, and the corresponding affinities were assessed through the BC50 parameter, a binding descriptor univocally defining the affinity of a host for a guest in multi-equilibrium systems. Results show that the tripodal catecholic receptor binds the octyl glycosides with affinities ranging from 0.87 to 5.2 mM and with a 6-fold selectivity factor for the alpha-mannoside over the beta-glucoside. Although the affinity for glycosides was not appreciably improved with respect to the ureidic receptor, a significant change in selectivity was obtained by the H-bonding group replacement.

  1. Measurement of the solar constant

    NASA Technical Reports Server (NTRS)

    Crommelynck, D.

    1981-01-01

    The absolute value of the solar constant and the long term variations that exist in the absolute value of the solar constant were measured. The solar constant is the total irradiance of the Sun at a distance of one astronomical unit. An absolute radiometer removed from the effects of the atmosphere with its calibration tested in situ was used to measure the solar constant. The importance of an accurate knowledge of the solar constant is emphasized.

  2. Using NMR to Develop New Allosteric and Allo-Network Drugs.

    PubMed

    Smith, Robert E; Tran, Kevin; Richards, Kristy M; Luo, Rensheng

    2015-01-01

    NMR is becoming an important tool for developing new allosteric and allo-network drugs that bind to allosteric sites on enzymes, partially inhibiting them and causing fewer side effects than drugs already developed that target active sites. This is based on systems thinking, in which active enzymes and other proteins are known to be flexible and interact with each other. In other words, proteins can exist in an ensemble of different conformations whose populations are tunable. NMR is being used to find the pathways through which the effects of binding of an allosteric ligand propagate. There are NMR screening assays for studying ligand binding. This includes determining the changes in the spin lattice relaxation due to changes in the mobility of atoms involved in the binding, measuring magnetization transfer from the protein to the ligand by saturation difference transfer NMR (STD-NMR) and the transfer of bulk magnetization to the ligand by water-Ligand Observed via Gradient Spectroscopy, or waterLOGSY. The chemical shifts of (1)H and (15)N of some of the atoms in amino acids change when an allosteric ligand binds to a protein. So, (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra can be used to identify key amino acids and ligand binding sites. The NMR chemical shifts of amino acids affected by ligand binding form a network that can be characterized. Allosteric networks can be identified by chemical shift covariance analysis (CHESCA). This approach has been used recently to study the binding of new molecular entities (NMEs) to potentially therapeutic drug targets. PMID:26577663

  3. The Hubble constant.

    PubMed

    Tully, R B

    1993-06-01

    Five methods of estimating distances have demonstrated internal reproducibility at the level of 5-20% rms accuracy. The best of these are the cepheid (and RR Lyrae), planetary nebulae, and surface-brightness fluctuation techniques. Luminosity-line width and Dn-sigma methods are less accurate for an individual case but can be applied to large numbers of galaxies. The agreement is excellent between these five procedures. It is determined that Hubble constant H0 = 90 +/- 10 km.s-1.Mpc-1 [1 parsec (pc) = 3.09 x 10(16) m]. It is difficult to reconcile this value with the preferred world model even in the low-density case. The standard model with Omega = 1 may be excluded unless there is something totally misunderstood about the foundation of the distance scale or the ages of stars. PMID:11607391

  4. When constants are important

    SciTech Connect

    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.

  5. The Hubble constant.

    PubMed Central

    Tully, R B

    1993-01-01

    Five methods of estimating distances have demonstrated internal reproducibility at the level of 5-20% rms accuracy. The best of these are the cepheid (and RR Lyrae), planetary nebulae, and surface-brightness fluctuation techniques. Luminosity-line width and Dn-sigma methods are less accurate for an individual case but can be applied to large numbers of galaxies. The agreement is excellent between these five procedures. It is determined that Hubble constant H0 = 90 +/- 10 km.s-1.Mpc-1 [1 parsec (pc) = 3.09 x 10(16) m]. It is difficult to reconcile this value with the preferred world model even in the low-density case. The standard model with Omega = 1 may be excluded unless there is something totally misunderstood about the foundation of the distance scale or the ages of stars. PMID:11607391

  6. Unitaxial constant velocity microactuator

    DOEpatents

    McIntyre, Timothy J.

    1994-01-01

    A uniaxial drive system or microactuator capable of operating in an ultra-high vacuum environment. The mechanism includes a flexible coupling having a bore therethrough, and two clamp/pusher assemblies mounted in axial ends of the coupling. The clamp/pusher assemblies are energized by voltage-operated piezoelectrics therewithin to operatively engage the shaft and coupling causing the shaft to move along its rotational axis through the bore. The microactuator is capable of repeatably positioning to sub-manometer accuracy while affording a scan range in excess of 5 centimeters. Moreover, the microactuator generates smooth, constant velocity motion profiles while producing a drive thrust of greater than 10 pounds. The system is remotely controlled and piezoelectrically driven, hence minimal thermal loading, vibrational excitation, or outgassing is introduced to the operating environment.

  7. Unitaxial constant velocity microactuator

    DOEpatents

    McIntyre, T.J.

    1994-06-07

    A uniaxial drive system or microactuator capable of operating in an ultra-high vacuum environment is disclosed. The mechanism includes a flexible coupling having a bore therethrough, and two clamp/pusher assemblies mounted in axial ends of the coupling. The clamp/pusher assemblies are energized by voltage-operated piezoelectrics therewithin to operatively engage the shaft and coupling causing the shaft to move along its rotational axis through the bore. The microactuator is capable of repeatably positioning to sub-nanometer accuracy while affording a scan range in excess of 5 centimeters. Moreover, the microactuator generates smooth, constant velocity motion profiles while producing a drive thrust of greater than 10 pounds. The system is remotely controlled and piezoelectrically driven, hence minimal thermal loading, vibrational excitation, or outgassing is introduced to the operating environment. 10 figs.

  8. Constant attitude orbit transfer

    NASA Astrophysics Data System (ADS)

    Cress, Peter; Evans, Michael

    A two-impulse orbital transfer technique is described in which the spacecraft attitude remains constant for both burns, eliminating the need for attitude maneuvers between the burns. This can lead to significant savings in vehicle weight, cost and complexity. Analysis is provided for a restricted class of applications of this transfer between circular orbits. For those transfers with a plane change less than 30 deg, the total velocity cost of the maneuver is less than twelve percent greater than that of an optimum plane split Hohmann transfer. While this maneuver does not minimize velocity requirement, it does provide a means of achieving necessary transfer while substantially reducing the cost and complexity of the spacecraft.

  9. A Constant Pressure Bomb

    NASA Technical Reports Server (NTRS)

    Stevens, F W

    1924-01-01

    This report describes a new optical method of unusual simplicity and of good accuracy suitable to study the kinetics of gaseous reactions. The device is the complement of the spherical bomb of constant volume, and extends the applicability of the relationship, pv=rt for gaseous equilibrium conditions, to the use of both factors p and v. The method substitutes for the mechanical complications of a manometer placed at some distance from the seat of reaction the possibility of allowing the radiant effects of reaction to record themselves directly upon a sensitive film. It is possible the device may be of use in the study of the photoelectric effects of radiation. The method makes possible a greater precision in the measurement of normal flame velocities than was previously possible. An approximate analysis shows that the increase of pressure and density ahead of the flame is negligible until the velocity of the flame approaches that of sound.

  10. Determination of fat content in NMR images of meat

    NASA Astrophysics Data System (ADS)

    Ballerini, Lucia

    2000-12-01

    In this paper we present an application to food science of image processing technique. We describe a method for determining fat content in beef meat. The industry of meat faces a permanent need for improved methods for meat quality evaluation. Researchers want improved techniques to deepen their understanding of meat features. Expectations of consumers for meat quality grow constantly, which induces the necessity of quality control. Recent advances in the area of computer and video processing have created new ways to monitor quality in the food industry. We investigate the use of a new technology to control the quality of food: NMR imaging. The inherent advantages of NMR images are many. Chief among these unprecedented contrasts between the various structures present in meat like muscle, fat, and connective tissue. Moreover, the three-dimensional nature of the NMR method allow us to analyze isolated cross-sectional slices of the meat and to measure the volumetric content of fat, not only the fat visible on the surface. We propose a segmentation algorithm for the detection of fat together with a filtering technique to remove intensity inhomogeneities in NMR images caused by non-uniformities of the magnetic field during acquisition. Measurements have been successfully correlated with chemical analysis and digital photography. Results show that the NMR technique is a promising non-invasive method to determine the fat content in meat.

  11. Programming A Molecular Relay for Ultrasensitive Biodetection through 129 Xe NMR

    SciTech Connect

    Wang, Yanfei; Roose, Benjamin W.; Philbin, John P.; Doman, Jordan L.; Dmochowski, Ivan J.

    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.

  12. Whole-core analysis by sup 13 C NMR

    SciTech Connect

    Vinegar, H.J.; Tutunjian, P.N. ); Edelstein, W.A.; Roemer, P.B. )

    1991-06-01

    This paper reports on a whole-core nuclear magnetic resonance (NMR) system that was used to obtain natural abundance {sup 13}C spectra. The system enables rapid, nondestructive measurements of bulk volume of movable oil, aliphatic/aromatic ratio, oil viscosity, and organic vs. carbonate carbon. {sup 13}C NMR can be used in cores where the {sup 1}H NMR spectrum is too broad to resolve oil and water resonances separately. A 5 1/4-in. {sup 13}C/{sup 1}H NMR coil was installed on a General Electric (GE) CSI-2T NMR imager/spectrometer. With a 4-in.-OD whole core, good {sup 13}C signal/noise ratio (SNR) is obtained within minutes, while {sup 1}H spectra are obtained in seconds. NMR measurements have been made of the {sup 13}C and {sup 1}H density of crude oils with a wide range of API gravities. For light- and medium-gravity oils, the {sup 13}C and {sup 1}H signal per unit volume is constant within about 3.5%. For heavy crudes, the {sup 13}C and {sup 1}H density measured by NMR is reduced by the shortening of spin-spin relaxation time. {sup 13}C and {sup 1}H NMR spin-lattice relaxation times were measured on a suite of Cannon viscosity standards, crude oils (4 to 60{degrees} API), and alkanes (C{sub 5} through C{sub 16}) with viscosities at 77{degrees}F ranging from 0.5 cp to 2.5 {times} 10{sup 7} cp. The {sup 13}C and {sup 1}H relaxation times show a similar correlation with viscosity from which oil viscosity can be estimated accurately for viscosities up to 100 cp. The {sup 13}C surface relaxation rate for oils on water-wet rocks is very low. Nonproton decoupled {sup 13}C NMR is shown to be insensitive to kerogen; thus, {sup 13}C NMR measures only the movable hydrocarbon content of the cores. In carbonates, the {sup 13}C spectrum also contains a carbonate powder pattern useful in quantifying inorganic carbon and distinguishing organic from carbonate carbon.

  13. Sensitization of a stray-field NMR to vibrations: a potential for MR elastometry with a portable NMR sensor.

    PubMed

    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.

  14. Studies of electrolyte penetration in carbon anodes by NMR techniques.

    SciTech Connect

    Sandi, G.

    1998-12-09

    A toroid cavity nuclear magnetic resonance (NMR) detector capable of recording radial concentration profiles, diffusion constants, and displacements of charge carriers was employed to investigate the lithium ion distribution in an electrochemical cell containing a carbonaceous material synthesized from pyrene and pillared clays as inorganic templates. A carbon rod was used in a control experiment to assign the Li{sup +} spectrum and to calibrate the one dimensional radial images.

  15. The bulge region of HIV-1 TAR RNA binds metal ions in solution.

    PubMed

    Olejniczak, Mikołaj; Gdaniec, Zofia; Fischer, Artur; Grabarkiewicz, Tomasz; Bielecki, Lukasz; Adamiak, Ryszard W

    2002-10-01

    Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.9 +/- 0.6 and 2.7 +/- 1.7 mM, respectively. Argininamide, inducing largest (19)F chemical shifts changes at FU-23, was used as a reference ligand (K(d) = 0.3 +/- 0.1 mM). In the Pb2+-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg2+ and Ca2+ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb2+-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH3)6(3+) > Mg2+ > Ca2+] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.

  16. Compact orthogonal NMR field sensor

    SciTech Connect

    Gerald, II, Rex E.; Rathke, Jerome W.

    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.

  17. Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement.

    PubMed

    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.

  18. Synthesis, chemical characterization, DNA binding and antioxidant studies of ferrocene incorporated selenoure

    NASA Astrophysics Data System (ADS)

    Hussain, Raja Azadar; Badshah, Amin; Sohail, Manzar; Lal, Bhajan; Akbar, Kamran

    2013-09-01

    In this article we have reported synthesis, chemical characterization (with single crystal XRD, elemental analysis, FTIR and multinuclear NMR spectroscopy), DNA binding (with cyclic voltammetry, UV-vis spectroscopy, molecular docking and viscometry) and antioxidant activities (1,1-diphenyl-2-picrylhydrazyl scavenging) of 1-(2-methylbenzoyl)-3-(3-ferrocenylphenyl)selenourea (MOT). We found that this compound interacts electrostatically with DNA and has a binding constant value of 1.703 × 104 M-1. Lower value of diffusion coefficient for MOT-DNA adduct (1.35 × 106 cm2 s-1) relative to free MOT (1.66 × 106 cm2 s-1) in cyclic voltammetry (CV) indicated the binding of the compound with DNA. Smaller value of binding site size (0.88 base pairs) in CV, hyperchromism in UV-vis spectroscopy and decrease of relative specific viscosity of DNA in viscometry favored electrostatic interactions. Binding energy of experimental (-5.77 kcal mol-1) and simulated (-5.86 kcal mol-1) work are in close agreement with each other. IC50 value of MOT for 1,1-diphenyl-2-picrylhydrazyl scavenging was found to be 27 μM.

  19. Benzocaine complexation with p-sulfonic acid calix[n]arene: experimental ((1) H-NMR) and theoretical approaches.

    PubMed

    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.

  20. NMR characterization of thin films

    DOEpatents

    Gerald II, Rex E.; Klingler, Robert J.; Rathke, Jerome W.; Diaz, Rocio; Vukovic, Lela

    2010-06-15

    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.

  1. NMR characterization of thin films

    DOEpatents

    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.

  2. Optical pumping and xenon NMR

    SciTech Connect

    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.

  3. Optical pumping and xenon NMR

    SciTech Connect

    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.

  4. Expanding sapphyrin: towards selective phosphate binding.

    PubMed

    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.

  5. Automated protein NMR resonance assignments.

    PubMed

    Wan, Xiang; Xu, Dong; Slupsky, Carolyn M; Lin, Guohui

    2003-01-01

    NMR resonance peak assignment is one of the key steps in solving an NMR protein structure. The assignment process links resonance peaks to individual residues of the target protein sequence, providing the prerequisite for establishing intra- and inter-residue spatial relationships between atoms. The assignment process is tedious and time-consuming, which could take many weeks. Though there exist a number of computer programs to assist the assignment process, many NMR labs are still doing the assignments manually to ensure quality. This paper presents (1) a new scoring system for mapping spin systems to residues, (2) an automated adjacency information extraction procedure from NMR spectra, and (3) a very fast assignment algorithm based on our previous proposed greedy filtering method and a maximum matching algorithm to automate the assignment process. The computational tests on 70 instances of (pseudo) experimental NMR data of 14 proteins demonstrate that the new score scheme has much better discerning power with the aid of adjacency information between spin systems simulated across various NMR spectra. Typically, with automated extraction of adjacency information, our method achieves nearly complete assignments for most of the proteins. The experiment shows very promising perspective that the fast automated assignment algorithm together with the new score scheme and automated adjacency extraction may be ready for practical use. PMID:16452794

  6. Report on neptunium speciation by NMR and optical spectroscopies

    SciTech Connect

    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.

  7. 13C CP MAS NMR and GIAO-CHF calculations of coumarins.

    PubMed

    Zolek, Teresa; Paradowska, Katarzyna; Wawer, Iwona

    2003-01-01

    13C cross-polarization magic-angle spinning NMR spectra were recorded for a series of solid coumarins. Ab initio calculations of shielding constants were performed with the use of GIAO-CHF method. The combined CPMAS NMR and theoretical approach was successful in characterizing solid-state conformations of coumarins; a relationship sigma (ppm) = -1.032 xdelta + 205.28 (R(2) = 0.9845) can be used to obtain structural information for coumarins, for which solid-state NMR or crystal structure data are not available.

  8. New methods of structure refinement for macromolecular structure determination by NMR

    PubMed Central

    Clore, G. Marius; Gronenborn, Angela M.

    1998-01-01

    Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13C shifts, 1H shifts, T1/T2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters. PMID:9600889

  9. Beyond the Hubble Constant

    NASA Astrophysics Data System (ADS)

    1995-08-01

    about the distances to galaxies and thereby about the expansion rate of the Universe. A simple way to determine the distance to a remote galaxy is by measuring its redshift, calculate its velocity from the redshift and divide this by the Hubble constant, H0. For instance, the measured redshift of the parent galaxy of SN 1995K (0.478) yields a velocity of 116,000 km/sec, somewhat more than one-third of the speed of light (300,000 km/sec). From the universal expansion rate, described by the Hubble constant (H0 = 20 km/sec per million lightyears as found by some studies), this velocity would indicate a distance to the supernova and its parent galaxy of about 5,800 million lightyears. The explosion of the supernova would thus have taken place 5,800 million years ago, i.e. about 1,000 million years before the solar system was formed. However, such a simple calculation works only for relatively ``nearby'' objects, perhaps out to some hundred million lightyears. When we look much further into space, we also look far back in time and it is not excluded that the universal expansion rate, i.e. the Hubble constant, may have been different at earlier epochs. This means that unless we know the change of the Hubble constant with time, we cannot determine reliable distances of distant galaxies from their measured redshifts and velocities. At the same time, knowledge about such change or lack of the same will provide unique information about the time elapsed since the Universe began to expand (the ``Big Bang''), that is, the age of the Universe and also its ultimate fate. The Deceleration Parameter q0 Cosmologists are therefore eager to determine not only the current expansion rate (i.e., the Hubble constant, H0) but also its possible change with time (known as the deceleration parameter, q0). Although a highly accurate value of H0 has still not become available, increasing attention is now given to the observational determination of the second parameter, cf. also the Appendix at the

  10. α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF

    PubMed Central

    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. PMID:27584012

  11. α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF.

    PubMed

    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.

  12. α-Mangostin Extraction from the Native Mangosteen (Garcinia mangostana L.) and the Binding Mechanisms of α-Mangostin to HSA or TRF.

    PubMed

    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. PMID:27584012

  13. 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.

    PubMed Central

    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

  14. Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

    PubMed Central

    Linder, M.; Mattinen, M. L.; Kontteli, M.; Lindeberg, G.; Ståhlberg, J.; Drakenberg, T.; Reinikainen, T.; Pettersson, G.; Annila, A.

    1995-01-01

    Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly. PMID:7549870

  15. Medical applications of NMR imaging and NMR spectroscopy with stable isotopes. Summary

    SciTech Connect

    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.

  16. jsNMR: an embedded platform-independent NMR spectrum viewer.

    PubMed

    Vosegaard, Thomas

    2015-04-01

    jsNMR is a lightweight NMR spectrum viewer written in JavaScript/HyperText Markup Language (HTML), which provides a cross-platform spectrum visualizer that runs on all computer architectures including mobile devices. Experimental (and simulated) datasets are easily opened in jsNMR by (i) drag and drop on a jsNMR browser window, (ii) by preparing a jsNMR file from the jsNMR web site, or (iii) by mailing the raw data to the jsNMR web portal. jsNMR embeds the original data in the HTML file, so a jsNMR file is a self-transforming dataset that may be exported to various formats, e.g. comma-separated values. The main applications of jsNMR are to provide easy access to NMR data without the need for dedicated software installed and to provide the possibility to visualize NMR spectra on web sites.

  17. Proton NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D sub 9k

    SciTech Connect

    Koerdel, J.; Forsen, S.; Chazin, W.J. )

    1989-08-22

    A wide range of two-dimensional {sup 1}H NMR experiments have been used to completely assign the 500-MHz {sup 1}H NMR spectrum of recombinant Ca{sup 2+}-saturated bovine calbindin D{sub 9k}. In solution, calbindin D{sub 9k} exists as an equilibrium mixture of isoforms with trans (75%) and cis (25%) isomers of the peptide bond at Pro43 which results in two sets of {sup 1}H NMR signals from approximately half of the amino acids. The complete {sup 1}H NMR assignments for the major, trans-pro43 isoform are presented here. By use of an integrated strategy for spin system identification, 62 of the 76 spin systems could be assigned to the appropriate residue type. Sequence-specific assignments were then obtained by the standard method. Secondary structure elements were identified on the basis of networks of sequential and medium-range nuclear Overhauser effects (NOEs), {sup 3}J{sub HN{alpha}} spin coupling constants, and the location of slowly exchanging amide protons. Four helical segments and a short {beta}-sheet between the two calcium binding loops are found. These elements of secondary structure and a few additional long-range NOEs provide the global fold. Good agreement is found between the solution and crystal structures of the minor A form of bovine calbindin D{sub 9k} and between the solution structures of the minor A form of bovine calbindin D{sub 9k} and intact porcine calbindin D{sub 9k}.

  18. NMR planar microcoil for microanalysis

    NASA Astrophysics Data System (ADS)

    Sorli, B.; Chateaux, J. F.; Quiquerez, L.; Bouchet-Fakri, L.; Briguet, A.; Morin, P.

    2006-11-01

    This article deals with the analysis of small sample volume by using a planar microcoil and a micromachined cavity. This microcoil is used as a nuclear magnetic resonance (NMR) radio frequency detection coil in order to perform in vitro NMR analysis of the sample introduced into the microcavity. It is a real challenging task to develop microsystem for NMR spectrum extraction for smaller and smaller sample volume. Moreover, it is advantageous that these microsystems could be integrated in a Micro Total Analysing System (μ -TAS) as an analysing tool. In this paper, NMR theory, description, fabrication process and electrical characterization of planar microcoils receiver are described. Results obtained on NMR microspectroscopy experiments have been performed on water and ethanol, using a 1 mm diameter planar coil. This microcoil is tuned and matched at 85.13 MHz which is the Larmor frequency of proton in a 2 T magnetic field. This paper has been presented at “3e colloque interdisciplinaire en instrumentation (C2I 2004)”, École Normale Supérieure de Cachan, 29 30 janvier 2004.

  19. NMR characterization of shocked quartz

    SciTech Connect

    Boslough, M.B.; Cygan, R.T.; Assink, R.A.; Kirkpatrick, R.J.

    1994-03-01

    We have characterized experimentally and naturally-shocked quartz (both synthetic and natural samples) by solid state nuclear magnetic resonance (NMR) spectroscopy. Relaxation analysis of experimentally-shocked samples provides a means for quantitative characterization of the amorphous/disordered silica component NMR spectra demonstrate that magnetization in both the amorphous and crystalline components follows power-law behavior as a function of recycle time. This observation is consistent with the relaxation of nuclear spins by paramagnetic impurities. A fractal dimension can be extracted from the power-law exponent associated with each phase, and relative abundances can be extracted from integrated intensities of deconvolved peaks. NMR spectroscopy of naturally-shocked sandstone from Meteor Crater, Arizona (USA) led to the discovery of a new amorphous hydroxylated silica phase. Solid state NMR spectra of both experimentally and naturally shocked quartz were unexpectedly rich in microstructural information, especially when combined with relaxation analysis and cross-polarization studies. We suggest solid state NMR as a potentially useful tool for examining shock-induced microstructural changes in other inorganic compounds, with possible implications for shock processing of structural ceramics.

  20. Re-evaluation of the model-free analysis of fast internal motion in proteins using NMR relaxation.

    PubMed

    Frederick, Kendra King; Sharp, Kim A; Warischalk, Nicholas; Wand, A Joshua

    2008-09-25

    NMR spin relaxation retains a central role in the characterization of the fast internal motion of proteins and their complexes. Knowledge of the distribution and amplitude of the motion of amino acid side chains is critical for the interpretation of the dynamical proxy for the residual conformational entropy of proteins, which can potentially significantly contribute to the entropy of protein function. A popular treatment of NMR relaxation phenomena in macromolecules dissolved in liquids is the so-called model-free approach of Lipari and Szabo. The robustness of the mode-free approach has recently been strongly criticized and the remarkable range and structural context of the internal motion of proteins, characterized by such NMR relaxation techniques, attributed to artifacts arising from the model-free treatment, particularly with respect to the symmetry of the underlying motion. We develop an objective quantification of both spatial and temporal asymmetry of motion and re-examine the foundation of the model-free treatment. Concerns regarding the robustness of the model-free approach to asymmetric motion appear to be generally unwarranted. The generalized order parameter is robustly recovered. The sensitivity of the model-free treatment to asymmetric motion is restricted to the effective correlation time, which is by definition a normalized quantity and not a true time constant and therefore of much less interest in this context. With renewed confidence in the model-free approach, we then examine the microscopic distribution of side chain motion in the complex between calcium-saturated calmodulin and the calmodulin-binding domain of the endothelial nitric oxide synthase. Deuterium relaxation is used to characterize the motion of methyl groups in the complex. A remarkable range of Lipari-Szabo model-free generalized order parameters are seen with little correlation with basic structural parameters such as the depth of burial. These results are contrasted with the

  1. Paramagnetic NMR Relaxation and Molecular Mechanics Studies of Chloroperoxidase-Indole Complex: Insights into the Mechanism of Chloroperoxidase-Catalyzed Regioselective Oxidation of Indole

    PubMed Central

    Zhang, Rui; He, Qinghao; Chatfield, David; Wang, Xiaotang

    2013-01-01

    To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the structure of the CPO-indole complex was studied using NMR relaxation measurements and computational techniques. The dissociation constant (KD) of the CPO-indole complex was calculated to be approximately 21 mM. The distances (r) between protons of indole and the heme iron calculated from NMR relaxation measurements and molecular docking revealed that the pyrrole ring of indole is oriented toward the heme with its 2-H pointing directly at the heme iron. Both KD and r values are independent of pH in the range of 3.0–6.5. The stability and structure of the CPO-indole complex are also independent of the concentration of chloride/iodide ion. Molecular docking suggests the formation of a hydrogen bond between the N–H of indole and the carboxyl O of Glu 183 in the binding of indole to CPO. Simulated annealing of the CPO-indole complex using r values from NMR experiments as distance restraints reveals that the van der Waals interactions were much stronger than the Coulomb interactions in indole binding to CPO, indicating that the association of indole with CPO is primarily governed by hydrophobic rather than electrostatic interactions. This work provides the first experimental and theoretical evidence for the long-sought mechanism that leads to the “unexpected” regioselectivity of CPO-catalyzed oxidation of indole. The structure of the CPO-indole complex will serve as a lighthouse in guiding the design of CPO mutants with tailor-made activities for biotechnological applications. PMID:23634952

  2. New Quasar Studies Keep Fundamental Physical Constant Constant

    NASA Astrophysics Data System (ADS)

    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

  3. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    PubMed

    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.

  4. NMR study of stable radicals in the gas phase

    NASA Astrophysics Data System (ADS)

    Obynochny, A. A.; Maryasov, A. G.; Shakirov, M. M.; Grigoriev, I. A.

    1993-05-01

    The temperature dependence of the NMR spectrum of methyl-substituted nitroxyl radical of the imidazoline series has been studied. The NMR signal induced by radicals in the gas phase has been observed. A shift of the lines of the NMR spectrum in the gas phase according to the Curie law is observed which allows one to determine the value of the hfi constant of the protons of different racial groups. The hfi constant for methyl-substituted radical within experimental accuracy coincides with those measured by other methods in the liquid phase. In the absorbed phase of the samples under study, a substantial contribution is made by the volumetric susceptibility of the liquid film. The diamagnetic contribution to the magnetic susceptibility of the radical in the liquid state has been measured (in the film of 2 × 10 -6). When the thickness of the adsorbed film is small, the molecular exchange between the liquid and gas phases becomes noticeable, causing a corresponding additional shift of the lines. The gas-kinetic cross section for the radical (120 Å 2) has been estimated from the temperature dependence of the line width in the gas phase.

  5. A 13C-NMR study of azacryptand complexes.

    PubMed

    Wild, Aljoscha A C; Fennell, Kevin; Morgan, Grace G; Hewage, Chandralal M; Malthouse, J Paul G

    2014-09-28

    An azacryptand has been solubilised in aqueous media containing 50% (v/v) dimethyl sulphoxide. (13)C-NMR has been used to determine how the azacryptand is affected by zinc binding at pH 10. Using (13)C-NMR and (13)C-enriched bicarbonate we have been able to observe the formation of 4 different carbamate derivatives of the azacryptand at pH 10. The azacryptand was shown to solubilise zinc or cadmium at alkaline pHs. Two moles of zinc are bound per mole of azacryptand and this complex binds 1 mole of carbonate. By replacing the zinc with cadmium-113 we have shown that the (13)C-NMR signal of the (13)C-enriched carbon of the bound carbonate is split into two triplets at 2.2 °C. This shows that two cadmium complexes are formed and in each of these complexes the carbonate group is bound by two magnetically equivalent metal ions. It also demonstrates that these cadmium complexes are not in fast exchange. From temperature studies we show that in the zinc complexes both complexes are in fast exchange with each other but are in slow exchange with free bicarbonate. HOESY is used to determine the position of the carbonate carbon in the complex. The solution and crystal structures of the zinc-carbonate-azacryptand complexes are compared. PMID:25091182

  6. Protein structure determination with paramagnetic solid-state NMR spectroscopy.

    PubMed

    Sengupta, Ishita; Nadaud, Philippe S; Jaroniec, Christopher P

    2013-09-17

    Many structures of the proteins and protein assemblies that play central roles in fundamental biological processes and disease pathogenesis are not readily accessible via the conventional techniques of single-crystal X-ray diffraction and solution-state nuclear magnetic resonance (NMR). On the other hand, many of these challenging biological systems are suitable targets for atomic-level structural and dynamic analysis by magic-angle spinning (MAS) solid-state NMR spectroscopy, a technique that has far less stringent limitations on the molecular size and crystalline state. Over the past decade, major advances in instrumentation and methodology have prompted rapid growth in the field of biological solid-state NMR. However, despite this progress, one challenge for the elucidation of three-dimensional (3D) protein structures via conventional MAS NMR methods is the relative lack of long-distance data. Specifically, extracting unambiguous interatomic distance restraints larger than ∼5 Å from through-space magnetic dipole-dipole couplings among the protein (1)H, (13)C, and (15)N nuclei has proven to be a considerable challenge for researchers. It is possible to circumvent this problem by extending the structural studies to include several analogs of the protein of interest, intentionally modified to contain covalently attached paramagnetic tags at selected sites. In these paramagnetic proteins, the hyperfine couplings between the nuclei and unpaired electrons can manifest themselves in NMR spectra in the form of relaxation enhancements of the nuclear spins that depend on the electron-nucleus distance. These effects can be significant for nuclei located up to ∼20 Å away from the paramagnetic center. In this Account, we discuss MAS NMR structural studies of nitroxide and EDTA-Cu(2+) labeled variants of a model 56 amino acid globular protein, B1 immunoglobulin-binding domain of protein G (GB1), in the microcrystalline solid phase. We used a set of six EDTA-Cu(2

  7. Advanced NMR characterization of zeolite catalysts

    NASA Astrophysics Data System (ADS)

    Welsh, L. B.

    1985-04-01

    The program discussed in this report is a two-year two-phase joint UOP-University of Illinois study of the application of improved high resolution solid state nuclear magnetic resonance (NMR) techniques to the characterization of zeolite catalysts. During the first phase of this program very pure, and in some cases isotopically enriched faujasites will be prepared and studied by magic angle sample spinning NMR (MASS NMR) and variable engine sample spinning NMR (VASS NMR) on 500 and 360 MHz (proton frequency) NMR spectrometers. The NMR techniques that will be emphasized are the measurement and analysis of the (17)O NMR properties, (27)Al NMR intensity quantitation, and (27)Al and (29)Si NMR relaxation rates. During the second phase of this program these NMR techniques will be used to study the effects of impurity concentration, dealumination treatments and cation exchange on the NMR properties of faujasites. The initial emphasis of this program during Phase I is on the preparation and measurement of the NMR properties of (17)O enriched Na-Y faujasties.

  8. Exploring the DNA binding mode of transition metal based biologically active compounds

    NASA Astrophysics Data System (ADS)

    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.

  9. On the Theory of Noncovalent Binding

    PubMed Central

    Mihailescu, Mihail; Gilson, Michael K.

    2004-01-01

    It is widely accepted that the binding constant of a receptor and ligand can be written as a two-body integral involving the interaction energy of the receptor and the ligand. Interestingly, however, three different theories of binding in the literature dictate three distinct integrals. The present study uses theory, as well as simulations of binding experiments, to test the validity of the three integrals. When binding is measured by a signal that detects the ligand in the binding site, the most accurate results are obtained by an integral of the Boltzmann factor, where the bound complex is defined in terms of an exclusive binding region. A novel prediction of this approach, that expanding a ligand can increase its binding constant, is borne out by the simulations. The simulations also show that abnormal binding isotherms can be obtained when the region over which the signal is detected deviates markedly from the exclusion zone. Interestingly, the binding constant measured by equilibrium dialysis, rather than by monitoring a localized signal, can yield a binding constant that differs from that obtained from a signal measurement, and that is matched best by the integral of the Mayer factor. PMID:15240441

  10. Enhanced Diffusion in a Polymer-Silica Nanocomposite Viewed by Pulse Field Gradient NMR

    NASA Astrophysics Data System (ADS)

    Zhong, Junyan; Wen, Wen-Yang; Jones, Alan A.

    2003-03-01

    Enhanced Diffusion in a Polymer-Silica Nanocomposite Viewed by Pulse Field Gradient NMR Junyan Zhong, Wen-Yang Wen and Alan A. Jones Carlson School of Chemistry and Biochemistry, Clark University, Worcester MA 01610 The addition of fumed silica to the high permeability random copolymer of tetrafluoroethylene (TFE) and 2,2-bis(trifluoromethyl)-4,5-difluoro-1,3-dioxole (PDD) increases diffusion by up to an order of magnitude. The self-diffusion constant of pentane was measured using pulse field gradient NMR and non-Fickian diffusion is observed in cast films of the pure polymer and nanocomposites containing 10, 20 and 30 wtNMR experiment, diffusion can be monitored for different periods of time with an apparent diffusion constant determined for each period. At all compositions, the apparent diffusion constant appears to be faster when observed over shorter times and decreases towards a plateau value at longer times. This result is indicative of tortuous diffusion. The addition of fumed silica not only increases the apparent self-diffusion constants but also changes the dependence of the apparent self-diffusion constants on the time over which diffusion occurs. The self-diffusion constants decrease more slowly as a function of observation time indicating better connectivity of the more permeable domains. The apparent diffusion constants also increase as a function of time after the introduction of pentane. This phenomenon is observed in the pure polymer as well and is described as conditioning of the membrane.

  11. (129)Xe NMR Relaxation-Based Macromolecular Sensing.

    PubMed

    Gomes, Muller D; Dao, Phuong; Jeong, Keunhong; Slack, Clancy C; Vassiliou, Christophoros C; Finbloom, Joel A; Francis, Matthew B; Wemmer, David E; Pines, Alexander

    2016-08-10

    We report a (129)Xe NMR relaxation-based sensing approach that exploits changes in the bulk xenon relaxation rate induced by slowed tumbling of a cryptophane-based sensor upon target binding. The amplification afforded by detection of the bulk dissolved xenon allows sensitive detection of targets. The sensor comprises a xenon-binding cryptophane cage, a target interaction element, and a metal chelating agent. Xenon associated with the target-bound cryptophane cage is rapidly relaxed and then detected after exchange with the bulk. Here we show that large macromolecular targets increase the rotational correlation time of xenon, increasing its relaxation rate. Upon binding of a biotin-containing sensor to avidin at 1.5 μM concentration, the free xenon T2 is reduced by a factor of 4. PMID:27472048

  12. Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant.

    PubMed

    Song, Jikui; Markley, John L

    2003-05-13

    Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites. The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor. The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site. Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 [Ardelt, W., and Laskowski, M., Jr. (1991) J. Mol. Biol. 220, 1041-1052]: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15. What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3. Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results. Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*). Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from

  13. Protein inhibitors of serine proteinases: role of backbone structure and dynamics in controlling the hydrolysis constant.

    PubMed

    Song, Jikui; Markley, John L

    2003-05-13

    Standard mechanism protein inhibitors of serine proteinases bind as substrates and are cleaved by cognate proteinases at their reactive sites. The hydrolysis constant for this cleavage reaction at the P(1)-P(1)' peptide bond (K(hyd)) is determined by the relative concentrations at equilibrium of the "intact" (uncleaved, I) and "modified" (reactive site cleaved, I*) forms of the inhibitor. The pH dependence of K(hyd) can be explained in terms of a pH-independent term, K(hyd) degrees, plus the proton dissociation constants of the newly formed amino and carboxylate groups at the cleavage site. Two protein inhibitors that differ from one another by a single residue substitution have been found to have K(hyd) degrees values that differ by a factor of 5 [Ardelt, W., and Laskowski, M., Jr. (1991) J. Mol. Biol. 220, 1041-1052]: turkey ovomucoid third domain (OMTKY3) has K(hyd) degrees = 1.0, and Indian peafowl ovomucoid third domain (OMIPF3), which differs from OMTKY3 by the substitution P(2)'-Tyr(20)His, has K(hyd) degrees = 5.15. What mechanism is responsible for this small difference? Is it structural (enthalpic) or dynamic (entropic)? Does the mutation affect the free energy of the I state, the I* state, or both? We have addressed these questions through NMR investigations of the I and I forms of OMTKY3 and OMIPF3. Information about structure was derived from measurements of NMR chemical shift changes and trans-hydrogen-bond J-couplings; information about dynamics was obtained through measurements of (15)N relaxation rates and (1)H-(15)N heteronuclear NOEs with model-free analysis of the results. Although the I forms of each variant are more dynamic than the corresponding I forms, the study revealed no appreciable difference in the backbone dynamics of either intact inhibitor (OMIPF3 vs OMTKY3) or modified inhibitor (OMIPF3* vs OMTKY3*). Instead, changes in chemical shifts and trans-hydrogen-bond J-couplings suggested that the K(hyd) degrees difference arises from

  14. Constant-Pressure Hydraulic Pump

    NASA Technical Reports Server (NTRS)

    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.

  15. "Solvent Effects" in 1H NMR Spectroscopy.

    ERIC Educational Resources Information Center

    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)

  16. Push-through Direction Injectin NMR Automation

    EPA Science Inventory

    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. Petrophysical applications of NMR imaging

    SciTech Connect

    Rothwell, W.P.; Vinegar, H.J.

    1985-12-01

    A system for obtaining high-resolution NMR images of oil field cores is described. Separate proton density and T/sub 2/ relaxation images are obtained to distinguish spatial variations of fluid-filled porosity and the physical nature of the pores. Results are presented for typical sandstones.

  18. Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR.

    PubMed

    Balzan, Riccardo; Fernandes, Laetitia; Comment, Arnaud; Pidial, Laetitia; Tavitian, Bertrand; Vasos, Paul R

    2016-01-01

    The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR. PMID:26967906