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

    PubMed

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

    2005-05-30

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

  3. 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.

  4. 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.

  5. Oxygen binding constants for human hemoglobin tetramers.

    PubMed

    Gill, S J; Di Cera, E; Doyle, M L; Bishop, G A; Robert, C H

    1987-06-30

    High-precision studies of oxygen binding in hemoglobin (HbA0) solutions at near-physiological concentrations (2-12 mM heme; pHs 7.0-9.1; various buffers) have led to an unanticipated result: an unmeasurably low contribution from the triply ligated species. We have obtained this result from new differential oxygen-binding measurements for human hemoglobin through the use of a thin-layer apparatus, which enables study of solutions at high Hb concentrations. The effect of tetramer dissociation into dimers, which becomes significant at hemoglobin concentrations below 1 mM in heme, is avoided. The analysis of the binding reactions is thus cast in terms of tetramer-binding polynomial written with overall Adair equilibrium constants which directly reflect the contributions of intermediate ligated species. The unmeasurable contribution of the triply ligated species renders the equilibrium constants of the third and fourth stepwise reactions practically undeterminable.

  6. Calculation of NMR Spin-Spin Coupling Constants in Strychnine.

    PubMed

    Helgaker, Trygve; Jaszuński, Michał; Świder, Paweł

    2016-11-18

    We compare the NMR indirect nuclear spin-spin coupling constants in strychnine calculated using density functional theory (DFT) with the semiempirical relativistic force field (RFF) method of Kutateladze and Mukhina (KM) (J. Org. Chem. 2015, 80, 10838-10848). DFT values significantly more accurate than those obtained by KM for their comparison with RFF values can be obtained, at a lower cost, by an appropriate selection of basis set.

  7. 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.

  8. Spin-rotation and NMR shielding constants in HCl

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    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 1H35Cl are CCl = -53.914 kHz and CH = 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.

  9. 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.

  10. NMR Study of Organic Counterion Binding to Perfluorinated Micellar Structures

    NASA Astrophysics Data System (ADS)

    Bossev, Dobrin; Matsumoto, Mustuo; Nakahara, Masaru

    2008-03-01

    In this study we have applied our previously developed NMR method to study the adsorption of tetramethylammonium (TMA^+) and tetraethylammonium (TEA^+) counterions to micelles formed by perfluorooctylsulfonate (FOS^-) surfactant in water at 30 C. These two counterions induce formation of threadlike surfactant structures that result in well pronounced viscoelastic properties of the solution. To selectively probe the degree of counterion binding we have used ^1H and ^19F NMR chemical shifts and self-diffusion coefficients that are sensitive to the Stern and diffuse double layers, respectively. The competitive adsorption of TMA^+ and TEA^+ was examined as a function of the TMA^+/TEA^+ ratio at a constant FOS^- concentration of 100 mM. The two counterions were found to form Stern layer around the FOS^- micelles with comparable packing; about one counterion per two micellized FOS molecules. When mixed at intermediate proportions, however, the TEA^+ counterion shows preferential binding; the concentration of TEA^+ in the Stern layer is found to be twice higher than that of TMA^+ at equal total respective concentrations in the solution. These results are discussed in terms of counterion size and hydrophobicity and presented in parallel with those that involved the smaller and more hydrophilic lithium counterion.

  11. 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)

  12. 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)

  13. Factors affecting the threading of axle molecules through macrocycles: Binding constants for semirotaxane formation

    PubMed Central

    Clifford, Thomas; Abushamleh, Ahmad; Busch, Daryle H.

    2002-01-01

    The threading of more or less linear axle molecules through macrocyclic molecules, a fundamental process relating to the formation of interlocked molecular structures, has been investigated through the study in acetone of the equilibrium constants for the formation of pseudorotaxanes by NMR methods. The 30 new axle molecules have in common a secondary ammonium group, present as the thiocyanate salt, and an anthracen-9-ylmethyl group, but are rendered unique by the second amine substituent. All rotaxanes involve the well known polyether macrocycle, benzo[24]crown-8. The constants for the binding of axles having linear groups ranging from 2 to 18 carbon atoms show little variation in binding constant but are divided into two groups by their equilibration rates. Those with less than five methylene groups react rapidly on the NMR timescale, whereas those having more than five methylene groups are slow. Branching inhibits binding, but the effect decreases as the branch is moved away from the amine. Phenyl groups weaken binding when close to the amine but strengthen binding when more remote. Some functional groups decrease pseudorotaxane stability (alcohol functions), whereas others increase binding (carboxylic acid groups). PMID:11959934

  14. Detection of site-specific binding and co-binding of ligands to macromolecules using sup 19 F NMR

    SciTech Connect

    Jenkins, B.G. )

    1991-01-01

    Study of ligand-macromolecular interactions by {sup 19}F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of {sup 19}F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple {sup 19}F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of {sup 19}F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed.

  15. Mg NMR in DNA solutions: Dominance of site binding effects.

    PubMed

    Rose, D M; Bleam, M L; Record, M T; Bryant, R G

    1980-11-01

    (25)Mg NMR spectroscopy is applied to a study of magnesium ion interactions with DNA, which is considered as a model for a linear polyelectrolyte. It is demonstrated that the magnesium ion spectrum is complicated by a non-Lorent-zian line shape and is dominated by the effects of chemical exchange with macromolecule binding sites. A distinction is made between specific-site interactions in which the magnesium ion loses a water molecule from the first coordination sphere on binding and those interactions, referred to as territorial binding, in which the ion maintains its first coordination sphere complement of solvent. The first type of site-binding interactions are shown to dominate the magnesium ion NMR spectrum, based on a consideration of the magnitudes of the observed (25)Mg relaxation rates compared with (23)Na relaxation rates, the clear contributions of chemical exchange-limited relaxation, and an ion displacement experiment employing sodium.

  16. 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.

  17. 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.

  18. NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

    PubMed Central

    Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

    2009-01-01

    The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

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

    PubMed

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

    2014-05-19

    Owing to its imidazole side chain, histidine participates in various processes such as enzyme catalysis, pH regulation, metal binding, and phosphorylation. The determination of exchange rates of labile protons for such a system is important for understanding its functions. However, these rates are too fast to be measured directly in an aqueous solution by using NMR spectroscopy. We have obtained the exchange rates of the NH3(+) amino protons and the labile NH(ε2) and NH(δ1) protons of the imidazole ring by indirect detection through nitrogen-15 as a function of temperature (272 Kconstants (1)J(N,H(i)) could also be determined accurately.

  20. Binding analysis between L-histidine immobilized and oligonucleotides by SPR and NMR.

    PubMed

    Cruz, Carla; Santos, Sandra D; Cabrita, Eurico J; Queiroz, João A

    2013-05-01

    Saturation transfer difference (STD) NMR technique and surface plasmon resonance (SPR) are used to study amino acid affinity supports-nucleotides interactions with L-histidine amino acid immobilized on a surface as model support. We have immobilized L-histidine ligand on a carboxymethyldextran-modified gold surface intended for surface plasmon resonance and we analyze the binding profiles of synthetic polynucleotides (1-6 base, sugar and backbone) by determining the equilibrium dissociation constant (KD). The SPR binding profile (square-shaped) is identical for all the complexes and the highest binding affinity can be found for polyA₆ followed by polyG₆. As expected, the 5'-mononucleotides have the lowest affinity. To further study the structural aspects of the interaction we investigate the polynucleotide binding preferences to L-histidine chromatography support by STD-NMR spectroscopy. These results revealed that an increase in the number of bases and backbone to 6 units leads to more contacts with the support, where the main driving force for the interaction with polynucleotides are through the base, except for polyC₆, which is mainly through sugar-phosphate backbone. Therefore, the combination of SPR measurements with STD-NMR technique allowed to establish fine details of the molecular recognition process involved in amino acid affinity supports-nucleotides complexes.

  1. Determination of Relative Counterion Binding Constant to Cationic Micelles.

    PubMed

    Khalid, Khalisanni; Noh, Muhammad Azri Mohd; Zain, Sharifuddin Md; Khan, M Niyaz

    2017-04-01

    The efficiency of counterion affinity towards ionic micelles is often described in terms of the degree of counterion binding (β X ) to ionic micelles or the conventional ion-exchange constant ([Formula: see text]) or relative binding constant ([Formula: see text]) of X (-) and Br(-) counterions. This review describes the use of ionized phenyl salicylate ions, PSa(-), as a new probe to determine [Formula: see text] values using a semiempirical spectrophotometric method. The value of [Formula: see text] is found to be comparable to reported values obtained using different probes by the semiempirical kinetic method as well as different physical methods. Application of semiempirical methods for calculation of [Formula: see text] or [Formula: see text] values involves an inherent assumption that these values are independent of the physicochemical characteristics of the probe molecule.

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  3. Binding mechanism of an SH3 domain studied by NMR and ITC.

    PubMed

    Demers, Jean-Philippe; Mittermaier, Anthony

    2009-04-01

    Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, DeltaH(D)(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, DeltaS(D)(30 degrees C) = 20.0 cal mol(-1) K(-1). The free protein and peptide have significantly higher heat capacity than the bound complex, DeltaC(p) = 352 cal mol(-1) K(-1), which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, DeltaH(D)(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of DeltaC(p). The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K(D), and range from k(on)(10 degrees C) = 1.03 x 10(8) M(-1) s(-1) to k(on)(50 degrees C) = 2.0 x 10

  4. Intracellular volume and apparent diffusion constants of perfused cancer cell cultures, as measured by NMR.

    PubMed

    Pilatus, U; Shim, H; Artemov, D; Davis, D; van Zijl, P C; Glickson, J D

    1997-06-01

    Diffusion NMR spectroscopy was used to study intracellular volume and apparent water diffusion constants in different cell lines (DU145, human prostate cancer; AT3, rat prostate cancer; MCF-7, human breast cancer; RIF-1, mouse fibrosacroma). The cells were grown on various matrices (collagen sponge, collagen beads, polystyrene beads) which enabled continuous growth in perfused high density cell culture suitable for NMR studies. In perfused cell systems, the attenuation of the water signal versus the squared gradient strength was fitted by the sum of two decaying exponentials. For the slowly decaying component the apparent water diffusion constant at 37 degrees C was 0.22 (+/-0.02) x 10(-9) s/m2 for all cell lines at diffusion times > 100 ms. It continuously increased up to 0.47 (+/-0.05) x 10(-9) s/m2 when the diffusion time was decreased to 8 ms, indicating restricted diffusion. No significant effect of the matrices was observed. The fractional volume of the slow component as determined from the biexponential diffusion curve correlated with the relative intracellular volume, as obtained from the cell density in the sample and the cell size as measured by light microscopy. Therefore, this simple NMR approach can be used to determine intracellular volume in perfused cell cultures suitable for NMR studies. Using this information in combination with spectroscopic data, changes in intracellular metabolite concentration can be detected even when the cellular volume is changing during the experiment. The apparent diffusion constant for the fast diffusing component varied with growth matrix, cell density and cell type and also showed the typical characteristics of restricted diffusion (increase of apparent diffusion constant with time).

  5. 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.

  6. Determination of the relative signs of J-coupling constants by 1D NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Jae-Seung; Lee, Soonchil

    2001-03-01

    A simple method of determining the relative signs of J-coupling constants between nuclear spins is introduced. The sign information is necessary for the exact operation of quantum gates using spin-spin couplings such as a Controlled-NOT gate. Once the magnitudes of J-coupling constants are known, 1D NMR experiment can reveal the relative signs by making a longitudinal three-spin order 4 Iz Sz Rz from Sz and observing 1D spectrum after 90^o pulse on I or R. The advantages of this new method over conventional methods determining the relative signs are that this method requires less severe selectivity on pulses than the ``selective decoupling'' or ``spin tickling'' experiments and that experiment time is much shorter than 2D NMR sequences like β-COSY. Using this method, we found that previously reported the signs of J(^13C, ^13C)'s in ^13C-labelled alanine in literatures is wrong, which has been used in 3-bit NMR quantum computing experiments.

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

    PubMed

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

    2016-12-28

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

  8. Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

    PubMed Central

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

    2012-01-01

    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 1H-15N Heteronuclear Single Quantum Coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 µM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC crosspeak 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. PMID:23146631

  9. 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.

  10. 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.

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

    PubMed

    Kutateladze, Andrei G; Mukhina, Olga A

    2014-09-05

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

  12. 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.

  13. Extracting Diffusion Constants from Echo-Time-Dependent PFG NMR Data Using Relaxation-Time Information

    NASA Astrophysics Data System (ADS)

    Vandusschoten, D.; Dejager, P. A.; Vanas, H.

    Heterogeneous (bio)systems are often characterized by several water-containing compartments that differ in relaxation time values and diffusion constants. Because of the relatively small differences among these diffusion constants, nonoptimal measuring conditions easily lead to the conclusion that a single diffusion constant suffices to describe the water mobility in a heterogeneous (bio)system. This paper demonstrates that the combination of a T2 measurement and diffusion measurements at various echo times (TE), based on the PFG MSE sequence, enables the accurate determination of diffusion constants which are less than a factor of 2 apart. This new method gives errors of the diffusion constant below 10% when two fractions are present, while the standard approach of a biexponential fit to the diffusion data in identical circumstances gives larger (>25%) errors. On application of this approach to water in apple parenchyma tissue, the diffusion constant of water in the vacuole of the cells ( D = 1.7 × 10 -9 m 2/s) can be distinguished from that of the cytoplasm ( D = 1.0 × 10 -9 m 2/s). Also, for mung bean seedlings, the cell size determined by PFG MSE measurements increased from 65 to 100 μm when the echo time increased from 150 to 900 ms, demonstrating that the interpretation of PFG SE data used to investigate cell sizes is strongly dependent on the T2 values of the fractions within the sample. Because relaxation times are used to discriminate the diffusion constants, we propose to name this approach diffusion analysis by relaxation- time- separated (DARTS) PFG NMR.

  14. Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

    PubMed Central

    Krishnamoorthy, Janarthanan; Yu, Victor C. K.; Mok, Yu-Keung

    2010-01-01

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

  15. Determination of the equilibrium constant of C60 fullerene binding with drug molecules.

    PubMed

    Mosunov, Andrei A; Pashkova, Irina S; Sidorova, Maria; Pronozin, Artem; Lantushenko, Anastasia O; Prylutskyy, Yuriy I; Parkinson, John A; Evstigneev, Maxim P

    2017-03-01

    We report a new analytical method that allows the determination of the magnitude of the equilibrium constant of complexation, Kh, of small molecules to C60 fullerene in aqueous solution. The developed method is based on the up-scaled model of C60 fullerene-ligand complexation and contains the full set of equations needed to fit titration datasets arising from different experimental methods (UV-Vis spectroscopy, (1)H NMR spectroscopy, diffusion ordered NMR spectroscopy, DLS). The up-scaled model takes into consideration the specificity of C60 fullerene aggregation in aqueous solution and allows the highly dispersed nature of C60 fullerene cluster distribution to be accounted for. It also takes into consideration the complexity of fullerene-ligand dynamic equilibrium in solution, formed by various types of self- and hetero-complexes. These features make the suggested method superior to standard Langmuir-type analysis, the approach used to date for obtaining quantitative information on ligand binding with different nanoparticles.

  16. 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.

  17. 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.

  18. Determination of rate constants of N-alkylation of primary amines by 1H NMR spectroscopy.

    PubMed

    Li, Chenghong

    2013-09-05

    Macromolecules containing N-diazeniumdiolates of secondary amines are proposed scaffolds for controlled nitrogen oxide (NO) release medical applications. Preparation of these compounds often involves converting primary amine groups to secondary amine groups through N-alkylation. However, N-alkylation results in not only secondary amines but tertiary amines as well. Only N-diazeniumdiolates of secondary amines are suitable for controlled NO release; therefore, the yield of secondary amines is crucial to the total NO load of the carrier. In this paper, (1)H NMR spectroscopy was used to estimate the rate constants for formation of secondary amine (k1) and tertiary amine (k2) for alkylation reagents such as propylene oxide (PO), methyl acrylate (MA), and acrylonitrile (ACN). At room temperature, the ratio of k2/k1 for the three reactions was found to be around 0.50, 0.026, and 0.0072.

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

    NASA Astrophysics Data System (ADS)

    Yoshii, Noriyuki; Okamura, Emiko

    2009-06-01

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

  20. Calcium binding by phosphatidylserine headgroups. Deuterium NMR study.

    PubMed Central

    Roux, M; Bloom, M

    1991-01-01

    The binding of calcium to headgroup deuterated 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) was investigated by using deuterium magnetic resonance in pure POPS membranes and in mixed 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS 5:1 (m:m) bilayers. Addition of CaCl2 to pure POPS bilayers led to two component spectra attributed, respectively, to liquid-crystallin POPS (less than 15 kHz) and POPS molecules in the calcium-induced dehydrated phase (cochleate) (approximately 120 kHz). The liquid-crystalline component has nearly disappeared at a Ca2+ to POPS ratio of 0.5, indicating that, under such conditions, most of the POPS molecules are in the precipitated cochleate phase. After dilution of the POPS molecules in zwitterionic POPC membranes (POPC/POPS 5:1 m:m), single component spectra characteristic of POPS in the liquid-crystalline state were observed in the presence of Molar concentrations of calcium ions (Ca2+ to POPS ratio greater than 50), showing that the amount of dehydrated cochleate PS-Ca2+ phase, if any, was low (less than 5%) under such conditions. Deuterium NMR data obtained in the 15-50 degrees C temperature range with the mixed PC/PS membranes, either in the absence or the presence of Ca2+ ions, indicate that the serine headgroup undergoes a temperature-induced conformational change, independent of the presence of Ca2+. This is discussed in relation to other headgroup perturbations such as that observed upon change of the membrane surface charge density. PMID:1883944

  1. Cadmium-substituted skeletal troponin C: cadmium-113 NMR spectroscopy and metal binding investigations

    SciTech Connect

    Ellis, P.D.; Strang, P.; Potter, J.D.

    1984-08-25

    The binding of cadmium to skeletal troponin C (STnC) has been measured in two ways. Equilibrium binding experiments have shown two cadmium binding sites on STnC with a high affinity for Cd/sup 2 +/ and two with a lower affinity for Cd/sup 2 +/. The former binding constant is comparable to Ca/sup 2 +/ binding to the Ca/sup 2 +/-Mg/sup 2 +/ (structural) sites of STnC; the latter is about a factor of one hundred less than Ca/sup 2 +/ binding to the Ca/sup 2 +/-specific (regulatory) sites of STnC. In the presence of Mg/sup 2 +/ the affinity of Cd/sup 2 +/ for the higher affinity sites was lowered. These data clearly suggest that the sites with high affinity for Cd/sup 2 +/ are the same as the Ca/sup 2 +/-Mg/sup 2 +/ sites. The /sup 113/Cd NMR is temperature-dependent. At room temperature two resonances at -107.8 and -112.7 ppm are seen. Lowering the temperature to 4/sup 0/C alters the cadmium exchange dynamics, and results in a four line /sup 113/Cd spectrum. The two new resonances probably arise from cadmium binding to the Ca/sup 2 +/-specific sites on STnC; whereas, the resonances at -107.8 and -112.7 ppm correspond to cadmium binding at the Ca/sup 2 +/-Mg/sup 2 +/-sites, respectively. When the /sup 113/Cd/sup 2 +/-substituted protein was titrated with Ca/sup 2 +/, the two resonances of the high affinity sites were reduced, followed by a reduction of the lower affinity Cd/sup 2 +/ sites. Since the direct binding experiments demonstrate that the high affinity Cd/sup 2 +/ sites are the Ca/sup 2 +/-Mg/sup 2 +/ sites, it is concerned that Cd/sup 2 +/ binding to the protein dramatically alters the affinity of the Ca/sup 2 +/-Mg/sup 2 +/ sites for Ca/sup 2 +/ possibly with allosteric coupling network between binding sites.

  2. Multi-site binding of epigallocatechin gallate to human serum albumin measured by NMR and isothermal titration calorimetry

    PubMed Central

    Eaton, Joshua D.

    2017-01-01

    The affinity of epigallocatechin gallate (EGCG) for human serum albumin (HSA) was measured in physiological conditions using NMR and isothermal titration calorimetry (ITC). NMR estimated the Ka (self-dissociation constant) of EGCG as 50 mM. NMR showed two binding events: strong (n1=1.8 ± 0.2; Kd1 =19 ± 12 μM) and weak (n2∼20; Kd2 =40 ± 20 mM). ITC also showed two binding events: strong (n1=2.5 ± 0.03; Kd1 =21.6 ± 4.0 μM) and weak (n2=9 ± 1; Kd2 =22 ± 4 mM). The two techniques are consistent, with an unexpectedly high number of bound EGCG. The strong binding is consistent with binding in the two Sudlow pockets. These results imply that almost all EGCG is transported in the blood bound to albumin and explains the wide tissue distribution and chemical stability of EGCG in vivo. PMID:28424370

  3. Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study.

    PubMed

    Hellmich, Ute A; Mönkemeyer, Leonie; Velamakanni, Saroj; van Veen, Hendrik W; Glaubitz, Clemens

    2015-12-01

    ABC transporters are fascinating examples of fine-tuned molecular machines that use the energy from ATP hydrolysis to translocate a multitude of substrates across biological membranes. While structural details have emerged on many members of this large protein superfamily, a number of functional details are still under debate. High resolution structures yield valuable insights into protein function, but it is the combination of structural, functional and dynamic insights that facilitates a complete understanding of the workings of their complex molecular mechanisms. NMR is a technique well-suited to investigate proteins in atomic resolution while taking their dynamic properties into account. It thus nicely complements other structural techniques, such as X-ray crystallography, that have contributed high-resolution data to the architectural understanding of ABC transporters. Here, we describe the heterologous expression of LmrA, an ABC exporter from Lactococcus lactis, in Escherichia coli. This allows for more flexible isotope labeling for nuclear magnetic resonance (NMR) studies and the easy study of LmrA's multidrug resistance phenotype. We use a combination of solid-state magic angle spinning (MAS) on the reconstituted transporter and solution NMR on its isolated nucleotide binding domain to investigate consequences of nucleotide binding to LmrA. We find that nucleotide binding affects the protein globally, but that NMR is also able to pinpoint local dynamic effects to specific residues, such as the Walker A motif's conserved lysine residue.

  4. 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.

  5. NMR shielding constants in PH3, absolute shielding scale, and the nuclear magnetic moment of 31P.

    PubMed

    Lantto, Perttu; Jackowski, Karol; Makulski, Włodzimierz; Olejniczak, Małgorzata; Jaszuński, Michał

    2011-09-29

    Ab initio values of the absolute shielding constants of phosphorus and hydrogen in PH(3) were determined, and their accuracy is discussed. In particular, we analyzed the relativistic corrections to nuclear magnetic resonance (NMR) shielding constants, comparing the constants computed using the four-component Dirac-Hartree-Fock approach, the four-component density functional theory (DFT), and the Breit-Pauli perturbation theory (BPPT) with nonrelativistic Hartree-Fock or DFT reference functions. For the equilibrium geometry, we obtained σ(P) = 624.309 ppm and σ(H) = 29.761 ppm. Resonance frequencies of both nuclei were measured in gas-phase NMR experiments, and the results were extrapolated to zero density to provide the frequency ratio for an isolated PH(3) molecule. This ratio, together with the computed shielding constants, was used to determine a new value of the nuclear magnetic dipole moment of (31)P: μ(P) = 1.1309246(50) μ(N).

  6. 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.

  7. Use of NMR Saturation Transfer Difference Spectroscopy to Study Ligand Binding to Membrane Proteins

    PubMed Central

    Venkitakrishnan, Rani Parvathy; Benard, Outhiriaradjou; Max, Marianna; Markley, John L.

    2013-01-01

    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 1H 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 1H NMR signals. 1H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the 1H 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. PMID:22976022

  8. Egg yolk identification and aging in mixed paint binding media by NMR spectroscopy.

    PubMed

    Sfakianaki, Sofia; Kouloumpi, Eleni; Anglos, Demetrios; Spyros, Apostolos

    2015-01-01

    NMR spectroscopy is a powerful analytical tool for the identification and quantitative analysis of organic materials in a cultural heritage context. In this report, we present an analytical NMR protocol for the identification and semiquantification of egg yolk binders and mixed binding media that also contain a drying oil, namely linseed oil. The samples studied have been artificially and/or naturally aged in order to simulate the composition of organic materials in paintings. Analysis of the 1D and 2D NMR spectra showed that egg yolk can be identified even in binding media of considerable age via signals originating from cholesterol and/or cholesterol oxidation products present in the aged binding medium. Based on cholesterol-related and other lipid signals in the NMR spectra of egg yolk binders, a molecular marker (R/F) that suggests the presence of egg yolk in paint binders is proposed. Via this marker, the presence of egg yolk in the organic material obtained from an early 18th century Greek icon is confirmed, and this is further verified by 2D NMR spectroscopy. It is demonstrated that NMR molecular markers developed to estimate the hydrolysis/oxidation state of oil paintings are also suitable for the analysis of egg yolk and mixed medium (egg yolk-linseed oil) binders, indicating the generality of the NMR methodological approach in the analysis of organic materials in a cultural heritage context. Copyright © 2014 John Wiley & Sons, Ltd.

  9. 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.

  10. Binding site identification and structure determination of protein-ligand complexes by NMR

    PubMed Central

    Ziarek, Joshua J.; Peterson, Francis C.; Lytle, Betsy L.; Volkman, Brian F.

    2013-01-01

    Over the last fifteen years, the role of NMR spectroscopy in the lead identification and optimization stages of pharmaceutical drug discovery has steadily increased. NMR occupies a unique niche in the biophysical analysis of drug-like compounds because of its ability to identify binding sites, affinities, and ligand poses at the level of individual amino acids without necessarily solving the structure of the protein-ligand complex. However, it can also provide structures of flexible proteins and low-affinity (Kd > 10-6 M) complexes, which often fail to crystallize. This article emphasizes a throughput-focused protocol that aims to identify practical aspects of binding site characterization, automated and semi-automated NMR assignment methods, and structure determination of protein-ligand complexes by NMR. PMID:21371594

  11. Dynamics and intramolecular ligand binding of DtxR studied by MD simulations and NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Yi, Myunggi; Bhattacharya, Nilakshee; Zhou, Huan-Xiang

    2005-11-01

    Diphtheria toxin repressor (DtxR) regulates the expression of the diphtheria toxin gene through intramolecular ligand binding (Wylie et al., Biochemistry 2005, 44:40-51). Protein dynamics is essential to the binding process of the Pro-rich (Pr) ligand to the C-terminal SH3 domain. We present MD and NMR results on the dynamics and ligand interactions of a Pr-SH3 construct of DtxR. NMR relaxation data (T1, T2, and NOE) showed that the Pr ligand is very flexible, suggesting that it undergoes binding/unbinding transitions. A 50-ns MD trajectory of the protein was used to calculate T1, T2, and NOE, reproducing the NMR results for the SH3 domain but not for the Pr segment. During the MD simulation, the ligand stayed bound to the SH3 domain; thus the simulation represented the bound state. The NMR data for the Pr-segment could be explained by assuming that they represented the average behavior of a fast binding/unbinding exchange. Though unbinding was not observed in the MD simulation, the simulation did show large fluctuations of a loop which forms part of the wall of the binding pocket. The fluctuations led to opening up of the binding pocket, thus weakening the interaction with the Pr segment and perhaps ultimately leading to ligand unbinding.

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

    PubMed

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

    2012-12-01

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

  13. 13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A.

    PubMed

    Bertini, I; Monnanni, R; Pellacani, G C; Sola, M; Vallee, B L; Auld, D S

    1988-01-01

    Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.

  14. New insights into glycopeptide antibiotic binding to cell wall precursors using SPR and NMR spectroscopy.

    PubMed

    Treviño, Juan; Bayón, Carlos; Ardá, Ana; Marinelli, Flavia; Gandolfi, Raffaella; Molinari, Francesco; Jimenez-Barbero, Jesús; Hernáiz, María J

    2014-06-10

    Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D-Ala-D-Ala C-terminus of peptidoglycan precursors. Vancomycin-resistant bacteria replace the dipeptide with the D-Ala-D-Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin-like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell-wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2KdAlaDAlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D-Ala and the D-Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKdAlaDAlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.

  15. Fluorescence and NMR investigations in the ligand binding properties of adenylate kinases

    SciTech Connect

    Reinstein, J.; Vetter, I.R.; Schlichting, I.; Roesch, P.; Wittinghofer, A.; Goody, R.S. )

    1990-08-14

    A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand {alpha},{omega}-di((3{prime} or 2{prime})-O-(N-methyl-anthraniloyl)adenosine-5{prime}) pentaphosphate (MAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 {times} 10{sup {minus}9} to 5 {times} 10{sup {minus}2} M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. Together with the results obtained by Tian et al on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.

  16. 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.

  17. Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP.

    PubMed

    Arai, Munehito; Ferreon, Josephine C; Wright, Peter E

    2012-02-29

    Determination of affinities and binding sites involved in protein-ligand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein-protein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1-10 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 × 10(10) M(-1) s(-1), approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions. © 2012 American Chemical Society

  18. Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

    SciTech Connect

    Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

    1985-07-25

    The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.

  19. 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.

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

    PubMed

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

    2016-07-15

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

  1. 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.

  2. 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.

  3. The Tubulin Binding Mode of MT Stabilizing and Destabilizing Agents Studied by NMR

    NASA Astrophysics Data System (ADS)

    Sánchez-Pedregal, Víctor M.; Griesinger, Christian

    Tubulin is a fascinating molecule that forms the cytoskeleton of the cells and plays an important role in cell division and trafficking of molecules. It polymerizes and depolymerizes in order to fulfill this biological function. This function can be modulated by small molecules that interfere with the polymerization or the depolymerization. In this article, the structural basis of this behavior is reviewed with special attention to the contribution of NMR spectroscopy. Complex structures of small molecules that bind to tubulin and microtubules will be discussed. Many of them have been determined using NMR spectroscopy, which proves to be an important method in tubulin research.

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

    PubMed

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

    2011-11-24

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

  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. A robust method for determining DNA binding constants using capillary zone electrophoresis.

    PubMed

    Li, C; Martin, L M

    1998-10-01

    Capillary zone electrophoresis (CZE or CE) with on-line UV detection was utilized to measure the binding constants between purified calf thymus DNA and a library of designed tetrapeptides which had been constructed using unnatural amino acids with thiazole ring side chains. Mixtures containing a constant amount of a tetrapeptide, the neutral marker (mesityl oxide), and varying concentrations of DNA were prepared and equilibrated at 8 degreesC for 12 h. CE was then utilized to separate unbound tetrapeptides from the DNA-peptide complex. The UV absorbance of the peak representing unbound tetrapeptide decreased incrementally as a result of increasing the concentration of DNA in the equilibrium mixture. The absorbance of the peak corresponding to the unbound tetrapeptide was obtained directly from the electropherogram and used in the calculation of the DNA-peptide binding constants. The binding constant for each tetrapeptide to calf thymus DNA was obtained from the negative slope of a Scatchard plot and a comparison of the binding constants for different peptides showed that the tetrapeptides in the library have DNA-binding affinities ranging from 10(2) to 10(6) M-1.

  8. 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.

  9. 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.

  10. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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.

  12. 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.

  13. Fluorescence and 19F NMR evidence that phenylalanine, 3-L-fluorophenylalanine and 4-L-fluorophenylalanine bind to the L-leucine specific receptor of Escherichia coli.

    PubMed Central

    Luck, L. A.; Johnson, C.

    2000-01-01

    The binding capacity of the L-leucine receptor from Escherichia coli was measured with L-phenylalanine and 4-fluoro-L-phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L-leucine, L-phenylalanine, and 4-fluoro-L-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein-induced shifts for the 4-fluoro-L-phenylalanine peak and 3-fluoro-L-phenylalanine when receptor is present. Evidence points to the binding of only the L-isomers of these fluorine analogs. PMID:11206079

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

    PubMed

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

    2010-11-01

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

  15. A combined NMR and computational approach to investigate peptide binding to a designed Armadillo repeat protein.

    PubMed

    Ewald, Christina; Christen, Martin T; Watson, Randall P; Mihajlovic, Maja; Zhou, Ting; Honegger, Annemarie; Plückthun, Andreas; Caflisch, Amedeo; Zerbe, Oliver

    2015-05-22

    The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20μs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design.

  16. The use of hammett constants to understand the non-covalent binding of aromatics.

    PubMed

    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.

  17. 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

  18. 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.

  19. The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships.

    PubMed

    Orsini, Michael J; Nesmelova, Irina; Young, Helen C; Hargittai, Balazs; Beavers, Mary Pat; Liu, Jingchun; Connolly, Peter J; Middleton, Steven A; Mayo, Kevin H

    2005-03-04

    Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.

  20. 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

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

    PubMed Central

    2011-01-01

    Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions. PMID:21569443

  2. 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

  3. 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.

  4. 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...

  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. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    SciTech Connect

    Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro; Kobayashi, Yuji; Kawahara, Kazuki; Yoshida, Takuya; Ohkubo, Tadayasu

    2015-12-04

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D{sub 2} (PGD{sub 2}) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP{sup +}, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  8. Phonon, IR, and Raman spectra, NMR parameters, and elastic constant calculations for AlH3 polymorphs.

    PubMed

    Vajeeston, P; Ravindran, P; Fjellvåg, H

    2011-10-06

    The electronic structure, lattice dynamics, and mechanical properties of AlH(3) phases have been studied by density functional calculations. The chemical bonding in different polymorphs of AlH(3) are evaluated on the basis of electronic structures, charge density analysis, and atomic charges, as well as bond overlap population analysis and the Born effective charges. The phonon dispersion relations and phonon density of states of all the polymorphs of AlH(3) are calculated by direct force-constant method. Application of pressure induces seqauence of phase transitions in β-AlH(3) which are understood from the phonon dispersive curves of the involved phases. The previously predicted phases (Chem. Mater. 2008, 20, 5997) are found to be dynamically stable. The calculated single crystal elastic constants reveal that all the studied AlH(3) polymorphs are easily compressible. The chemical bonding of these polymorphs have noticeable covalent character (except the hp2 phase) according to the present chemical bonding analyses. For all these polymorphs, the NMR-related parameters, such as isotropic chemical shielding, quadrupolar coupling constant, and quadrupolar asymmetry, are also calculated. All IR- and Raman-active phonon frequencies, as well as the corresponding intensities, are calculated for all the AlH(3) polymorphs and are compared with available experimental results.

  9. Theoretical study on conformation dynamics of three-station molecular shuttle in different environments and its influence on NMR chemical shifts and binding interactions.

    PubMed

    Liu, Pingying; Li, Wei; Liu, Li; Wang, Leyong; Ma, Jing

    2014-10-02

    Microscopic information on conformational flexibility and macrocycle-thread binding interactions is helpful in rational design of novel multistation molecular shuttles with interesting topology and functions. Molecular dynamics (MD) was applied to simulate conformational changes of thread and macrocycle of a three-station molecular shuttle in different chemical environments (vacuum, CD3CN-CDCl3 solution, and crystal). In contrast with the highly distorted thread conformation in the gas phase and nonpolar CDCl3 solution, the solvated thread in CD3CN-CDCl3 (1:1) mix solvents exhibited a relatively rigid structure. Experimental observations of preferential binding at the protonated dibenzylammonium group (station I) were rationalized by quantum chemical calculations of macrocycle-thread binding energies at three different stations. The orthogonality of site-specific binding interactions at three different stations was also revealed by the nearly constant binding energy obtained at each specific recognition center with the replacement of different neighboring groups and terminal stoppers on the thread. Conformational flexibility has little effect on NMR signals of binding sites, but for some protons that are close to the solvent molecules in the first solvent shell, their chemical shifts are sensitive to the local electrostatic environment caused by nearby solvents. In crystal, π stacking induced evident upfield shifts of NMR signals in comparison with the isolated monomer.

  10. Study of xenon binding in cryptophane-A using laser-induced NMR polarization enhancement

    SciTech Connect

    Luhmer, M.; Goodson, B.M.; Song, Y.Q.; Laws, D.D.; Kaiser, L.; Pines, A. |

    1999-04-14

    Xenon is chemically inert, yet exhibits NMR parameters that are highly sensitive to its chemical environment. Considerable work has therefore capitalized on the utility of {sup 129}Xe (I = 1/2) as a magnetic resonance probe of molecules, materials, and biological systems. In solution, spin-polarization transfer between laser-polarized xenon and the hydrogen nuclei of nearby molecules leads to signal enhancements in the resolved {sup 1}H NMR spectrum, offering new opportunities for probing the chemical environment of xenon atoms. Following binding of laser-polarized xenon to molecules of cryptophane-A, selective enhancements of the {sup 1}H NMR signals were observed. A theoretical framework for the interpretation of such experimental results is provided, and the spin polarization-induced nuclear Overhauser effects are shown to yield information about the molecular environment of xenon. The observed selective {sup 1}H enhancements allowed xenon-proton internuclear distances to be estimated. These distances reveal structural characteristics of the complex, including the preferred molecular conformations adopted by cryptophane-A upon binding of xenon.

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

    SciTech Connect

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

  12. Elucidation of the electronic structure of molecules with the help of NMR spin-spin coupling constants: the FH molecule.

    PubMed

    Gräfenstein, Jürgen; Tuttle, Tell; Cremer, Dieter

    2005-03-17

    It is demonstrated how the one-bond NMR spin-spin coupling constant (SSCC) (1)J(FH) can be used as a source of information on the electronic structure of the FH molecule. For this purpose, the best possible agreement between measured and calculated SSCC is achieved by large basis set coupled perturbed density functional theory calculations. Then, the calculated value is dissected into its four Ramsey terms: Fermi contact, the paramagnetic spin-orbit term, the diamagnetic spin-orbit term, and the spin dipole term, which in turn are decomposed into orbital contributions and then described by their spin densities and orbital current densities. In this way, the SSCC gives detailed information about the electronegativity of F, the bond polarity, the bond polarizability, the volume and the polarizability of sigma and pi lone pair orbitals, the s- or p-character of the bond orbital, the nature of the LUMO, and the density distribution around F.

  13. 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.

  14. 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.

  15. A new method for the determination of equilibrium constants through binding capacity measurements.

    PubMed

    Di Cera, E; Gill, S J

    1988-12-01

    The recent discovery of the negligible contribution of the triply ligated species to the oxygenation process of human hemoglobin A0 (S.J. Gill, E. Di Cera, M.L. Doyle, G.A. Bishop and C.H. Robert, Biochemistry 26 (1987) 3995) has pointed out the high precision of differential binding measurements. These measurements closely approximate the binding capacity (E. Di Cera, S.J. Gill and J. Wyman, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 449) of the system and can be used to calculate higher derivatives of the binding curve. We develop here a new method for the determination of equilibrium constants through binding capacity measurements by which the physical parameters expressing the optical properties of the system are eliminated in the data analysis.

  16. Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein.

    PubMed

    Santambrogio, C; Favretto, F; D'Onofrio, M; Assfalg, M; Grandori, R; Molinari, H

    2013-08-01

    Human liver fatty acid binding protein (hL-FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a 'head-in' or 'head-out' orientation of the carboxylate end. Electrospray-ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head-out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.

  17. 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.

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

    PubMed Central

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

    2016-01-01

    The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs. PMID:26677224

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

    PubMed

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

    2016-02-05

    The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. 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.

  1. Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

    PubMed Central

    Tavel, Laurette; Jaquillard, Lucie; Karsisiotis, Andreas I.; Saab, Fabienne; Jouvensal, Laurence; Brans, Alain; Delmas, Agnès F.; Schoentgen, Françoise; Cadene, Martine; Damblon, Christian

    2012-01-01

    Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1. PMID:22558375

  2. 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.

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

    SciTech Connect

    Roberts, Arthur G.; Diaz, M. Delores; Lampe, Jed N.; Shireman, Laura M.; Grinstead, Jeffrey S.; Dabrowski, Michael J.; Pearson, Josh T.; Bowman, Michael K.; Atkins, William M.; Campbell, A. Patricia

    2006-02-01

    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 μM, 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 Hill-equation-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 low-affinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energetic model is presented that combines the energetics

  4. Ligand-detected relaxation dispersion NMR spectroscopy: dynamics of preQ1-RNA binding.

    PubMed

    Moschen, Thomas; Wunderlich, Christoph Hermann; Spitzer, Romana; Levic, Jasmin; Micura, Ronald; Tollinger, Martin; Kreutz, Christoph

    2015-01-07

    An NMR-based approach to characterizing the binding kinetics of ligand molecules to biomolecules, like RNA or proteins, by ligand-detected Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments is described. A (15)N-modified preQ1 ligand is used to acquire relaxation dispersion experiments in the presence of low amounts of the Fsu class I preQ1 aptamer RNA, and increasing ligand concentrations to probe the RNA small molecule interaction. Our experimental data strongly support the conformational selection mechanism postulated. The approach gives direct access to two parameters of a ligand-receptor interaction: the off rate and the population of the small molecule-receptor complex. A detailed description of the kinetics underlying the ligand binding process is of crucial importance to fully understanding a riboswitch's function and to evaluate potential new antibiotics candidates targeting the noncoding RNA species. Ligand-detected NMR relaxation dispersion experiments represent a valuable diagnostic tool for the characterization of binding mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Steinmann, Casper; Bratholm, Lars Andersen; Olsen, Jógvan Magnus Haugaard; Kongsted, Jacob

    2017-02-14

    Full-protein nuclear magnetic resonance (NMR) shielding constants based on ab initio calculations are desirable, because they can assist in elucidating protein structures from NMR experiments. In this work, we present NMR shielding constants computed using a new automated fragmentation (J. Phys. Chem. B 2009, 113, 10380-10388) approach in the framework of polarizable embedding density functional theory. We extend our previous work to give both basis set recommendations and comment on how large the quantum mechanical region should be to successfully compute (13)C NMR shielding constants that are comparable with experiment. The introduction of a probabilistic linear regression model allows us to substantially reduce the number of snapshots that are needed to make comparisons with experiment. This approach is further improved by augmenting snapshot selection with chemical shift predictions by which we can obtain a representative subset of snapshots that gives the smallest predicted error, compared to experiment. Finally, we use this subset of snapshots to calculate the NMR shielding constants at the PE-KT3/pcSseg-2 level of theory for all atoms in the protein GB3.

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

    PubMed

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

    2016-11-21

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

  7. The Proline Enamine Formation Pathway Revisited in Dimethyl Sulfoxide: Rate Constants Determined via NMR.

    PubMed

    Haindl, Michael H; Hioe, Johnny; Gschwind, Ruth M

    2015-10-14

    Enamine catalysis is a fundamental activation mode in organocatalysis and can be successfully combined with other catalytic methods, e.g., photocatalysis. Recently, the elusive enamine intermediates were detected, and their stabilization modes were revealed. However, the formation pathway of this central organocatalytic intermediate is still a matter of dispute, and several mechanisms involving iminium and/or oxazolidinone are proposed. Here, the first experimentally determined rate constants and rates of enamine formation are presented using 1D selective exchange spectroscopy (EXSY) buildup curves and initial rate approximation. The trends of the enamine formation rates from exo-oxazolidinones and endo-oxazolidinones upon variation of the proline and water concentrations as well as the nucelophilic/basic properties of additives are investigated together with isomerization rates of the oxazolidinones. These first kinetic data of enamine formations in combination with theoretical calculations reveal the deprotonation of iminium intermediates as the dominant pathway in dimethyl sulfoxide (DMSO). The dominant enamine formation pathway varies according to the experimental conditions, e.g., the presence and strength of basic additives. The enamine formation is zero-order in proline and oxazolidinones, which excludes the direct deprotonation of oxazolidinones via E2 mechanism. The nucleophilicity of the additives influences only the isomerization rates of the oxazolidinones and not the enamine formation rates, which excludes a nucleophile-assisted anti elimination of oxazolidinones as a major enamine formation pathway.

  8. 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.

  9. NMR structure of DREAM: Implications for Ca(2+)-dependent DNA binding and protein dimerization.

    PubMed

    Lusin, Jacqueline D; Vanarotti, Murugendra; Li, Congmin; Valiveti, Aswani; Ames, James B

    2008-02-26

    DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.

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

    PubMed

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

    2017-02-14

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

  11. 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.

  12. NMR spectroscopic study of noble gas binding into the engineered cavity of HPr(I14A) from Staphylococcus carnosus.

    PubMed

    Nisius, Lydia; Stadler, Max; Kalbitzer, Hans Robert; Brunner, Eike

    2005-09-29

    Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, Delta, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.

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

    PubMed

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

    2014-08-01

    Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individual's blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. 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.

  15. The Use of Pseudo-equilibrium Constant Affords Improved QSAR Models of Human Plasma Protein Binding

    PubMed Central

    Zhu, Xiangwei; Sedykh, Alexander; Zhu, Hao; Liu, Shushen; Tropsha, Alexander

    2015-01-01

    Purpose To develop accurate in silico predictors of Plasma Protein Binding (PPB). Methods Experimental PPB data were compiled for over 1,200 compounds. Two endpoints have been considered: (1) fraction bound (%PPB); and (2) the logarithm of a pseudo binding constant (lnKa) derived from %PPB. The latter metric was employed because it reflects the PPB thermodynamics and the distribution of the transformed data is closer to normal. Quantitative Structure-Activity Relationship (QSAR) models were built with Dragon descriptors and three statistical methods. Results Five-fold external validation procedure resulted in models with the prediction accuracy (R2) of 0.67±0.04 and 0.66±0.04, respectively, and the mean absolute error (MAE) of 15.3±0.2% and 13.6±0.2%, respectively. Models were validated with two external datasets: 173 compounds from DrugBank, and 236 chemicals from the US EPA ToxCast project. Models built with lnKa were significantly more accurate (MAE of 6.2–10.7%) than those built with %PPB (MAE of 11.9–17.6%) for highly bound compounds both for the training and the external sets. Conclusion The pseudo binding constant (lnKa) is more appropriate for characterizing PPB binding than conventional %PPB. Validated QSAR models developed herein can be applied as reliable tools in early drug development and in chemical risk assessment. PMID:23568522

  16. Computational Approaches to the Chemical Equilibrium Constant in Protein-ligand Binding.

    PubMed

    Montalvo-Acosta, Joel José; Cecchini, Marco

    2016-12-01

    The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline.

  17. L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy

    PubMed Central

    Jonker, Hendrik R. A.; Ilin, Serge; Grimm, S. Kaspar; Wöhnert, Jens; Schwalbe, Harald

    2007-01-01

    Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding. PMID:17169991

  18. Protein NMR Studies of substrate binding to human blood group A and B glycosyltransferases.

    PubMed

    Peters, Thomas; Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica

    2017-03-03

    Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies have shown these enzymes to adopt an open conformation in the absence of substrates. Binding of either the donor substrate UDP-Gal, or of UDP induces a semi-closed conformation. In the presence of both, donor- and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical shift titrations of uniformly 2H,15N labeled GTA or GTB with UDP affected about 20% of all cross peaks in 1H,15N-TROSY-HSQC spectra reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical shift perturbation experiments using 1-13C-methyl Ile labeled samples revealed two Ile residues, Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY based relaxation dispersion experiments do not reveal s to ms time scale motions. Although this study reveals substantial conformational plasticity of GTA and GTB it remains enigmatic how binding of substrates shifts the enzymes into catalytically competent states.

  19. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay.

    PubMed

    Win, Maung Nyan; Klein, Joshua S; Smolke, Christina D

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with K(d) values of 2.50 and 4.00 microM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques.

  20. Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

    PubMed Central

    Win, Maung Nyan; Klein, Joshua S.; Smolke, Christina D.

    2006-01-01

    RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with Kd values of 2.50 and 4.00 μM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques. PMID:17038331

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

    SciTech Connect

    Cheng, Chih-Ming; Lee, Yuarn-Jang; Wang, Wei-Ting; Hsu, Chien-Ting; Tsai, Jing-Shin; Wu, Chien-Ming; Ou, Keng-Liang; and others

    2011-01-07

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

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

    PubMed

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

    2017-05-01

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

  3. 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.

  4. Human recombinant [C22A] FK506-binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR.

    PubMed Central

    Zhang, Z.; Li, W.; Logan, T. M.; Li, M.; Marshall, A. G.

    1997-01-01

    Hydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentation, combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry (MS). After a specified period of H/D exchange in solution, C22A FKBP was digested by pepsin under slow exchange conditions (pH 2.4, 0 degree C), and then subjected to on-line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independently by heteronuclear two-dimensional NMR on 15N-enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive the distributions of hydrogen exchange rate constants from the MS-determined deuterium exchange-in curves in either the holoprotein or its proteolytic segments. The MEM-derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate constants determined by NMR for individual amide protons. The rate constant distributions determined by both methods are consistent and complementary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins. PMID:9336843

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

    PubMed

    Schipper, Benjamin R; Ramstad, Tore

    2005-07-01

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

  6. Relative Binding Constants of Arsenical-Antidote Adducts Determined by NMR Spectroscopy

    DTIC Science & Technology

    1990-09-01

    2,3-dimercaptopropanol) and DMPS (2,3-dimercaptopropanesulfonic acid) had a higher affinity than DMSA (2,3-dimercaptosuccinic acid) for the two... DMSA and DMPS are less toxic analogues of BAL (4) which have polar moieties (carboxylic and sulfonic acids) that make them less lipophilic, more water... DMSA . The results presented below indicate that the relative stability of the various complexes depends upon the nature, the charge, and stereochemistry

  7. 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.

  8. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR

    SciTech Connect

    Tang, Chun; Schwieters, Charles D.; Clore, G. Marius

    2008-09-08

    Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation. Crystal structures have provided a static picture of the apo (usually open) and holo (usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography, NMR and other biophysical techniques. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to {mu}s regime) of a predominantly ({approx}95%) open form (represented by the apo crystal structure) and a minor ({approx}5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.

  9. On the acid dissociation constants of bilirubin and biliverdin. pKa values from 13C NMR spectroscopy.

    PubMed

    Lightner, D A; Holmes, D L; McDonagh, A F

    1996-02-02

    Biliverdin and bilirubin are naturally-occurring tetrapyrrolic bile pigments containing two propionic acid side chains. These side chains, and their propensity for ionization, are critical in the biological disposition of the pigments. Surprisingly, accurate dissociation constants for the propionic acid groups of biliverdin are unknown, and a wide range of values, extending over some 4 orders of magnitude, has been suggested for the Ka values of the propionic acid groups of bilirubin in aqueous solutions. Recently, pKa values of 6.7-9.3 have been reported for bilirubin--values much greater than the value of approximately 5 typical of propionic acid groups. These curiously high values, currently being used to explain the biological transport and metabolism of bilirubin and related compounds, have been attributed to intramolecular hydrogen bonding. We have determined the pKa values of 99% 13C-enriched (13CO2H) [8(3),12(3)-13C2]mesobilirubin-XIII, alpha, the corresponding biliverdin, and several monopropionic model compounds by 13C NMR spectroscopy. This technique allows direct observation and quantitative measurement of the carboxylic acid and carboxylate anion carbon signals. Analysis of the variation of carboxyl 13C NMR chemical shift with pH gave rubin pKa values of 4.2 and 4.9 and verdin pKa values of 3.9 and 5.3 in aqueous buffers containing only a very small quantity (0.086 mol fraction) of dimethyl sulfoxide. When extrapolated to water, the pKa values are essentially unchanged. The data provide the first experimentally-determined pKa values for a biliverdin. They indicate that intramolecular hydrogen bonding has little effect on the acid dissociation of bilirubin and suggest that the equilibrium acidity of the bilirubin carboxylic acid groups is not abnormally high but similar to the thermodynamic acidity found in other carboxylic acids, as originally suggested by Overbeek et al. (Overbeek, J. T. G., Vink, C. L. J., and Deenstra, H. (1955) Recl. Trav. Chim

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

    NASA Astrophysics Data System (ADS)

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

    2011-05-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Lemak, Alexander; Yee, Adelinda; Wu, Hong; Yap, Damian; Zeng, Hong; Dombrovski, Ludmila; Houliston, Scott; Aparicio, Samuel; Arrowsmith, Cheryl H

    2013-01-01

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

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

    PubMed

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

    2013-11-26

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

  14. [Determination of the binding constant of bovine serum albumin and gatifloxacin using affinity capillary electrophoresis].

    PubMed

    Yao, Zhi; Zhang, Haobo; Wu, Yi; Guo, Huaizhong

    2007-11-01

    The interaction between bovine serum albumin (BSA) and gatifloxacin (GT) was investigated using affinity capillary electrophoresis (ACE) method, mobility-shift method. The mobility ratio of the internal standard and the sample was used to calculate the binding constant K(b) of bovine serum albumin and gatifloxacin. At first, 20 mmol/L, pH 7.4 sodium phosphate buffer solution (PBS) added with 0 - 1 000 micromol/L GT as running buffer was used, BSA as the sample was used. K(b) calculated from this experiment was 4.4 x 10(4) L/mol. Then 0 - 12.5 micromol/L BSA was used as an additive to do the ACE experiment, the K(b) obtained was 4.2 x 10(4) L/mol. The two values matched well with each other. In order to improve the shape of the peak and restrain the adsorption of BSA to the internal wall of the quartz column, in the second method, 0.25 mol/L Gly and 0.5 mmol/L ethylenediamine tetracetate (EDTA) were added to the running buffer, and 0.5% sodium dodecyl sulfate (SDS) solution was used to rinse the column. Satisfactory effect was obtained. The K(b) from fluorescence method was also calculated with a value of 2.7 x 10(4) L/mol. This work demonstrated that the determination of the binding constant by ACE is simple with high performance.

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

    PubMed

    Bolívar, Beatriz E; Welch, John T

    2017-02-21

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

  16. Dissociation Rate Constants of Human Fibronectin Binding to Fibronectin-binding Proteins on Living Staphylococcus aureus Isolated from Clinical Patients*

    PubMed Central

    Casillas-Ituarte, Nadia N.; Lower, Brian H.; Lamlertthon, Supaporn; Fowler, Vance G.; Lower, Steven K.

    2012-01-01

    Staphylococcus aureus is part of the indigenous microbiota of humans. Sometimes, S. aureus bacteria enter the bloodstream, where they form infections on implanted cardiovascular devices. A critical, first step in such infections is a bond that forms between fibronectin-binding protein (FnBP) on S. aureus and host proteins, such as fibronectin (Fn), that coat the surface of implants in vivo. In this study, native FnBPs on living S. aureus were shown to form a mechanically strong conformational structure with Fn by atomic force microscopy. The tensile acuity of this bond was probed for 46 bloodstream isolates, each from a patient with a cardiovascular implant. By analyzing the force spectra with the worm-like chain model, we determined that the binding events were consistent with a multivalent, cluster bond consisting of ∼10 or ∼80 proteins in parallel. The dissociation rate constant (koff, s−1) of each multibond complex was determined by measuring strength as a function of the loading rate, normalized by the number of bonds. The bond lifetime (1/koff) was two times longer for bloodstream isolates from patients with an infected device (1.79 or 69.47 s for the 10- or 80-bond clusters, respectively; n = 26 isolates) relative to those from patients with an uninfected device (0.96 or 34.02 s; n = 20 isolates). This distinction could not be explained by different amounts of FnBP, as confirmed by Western blots. Rather, amino acid polymorphisms within the Fn-binding repeats of FnBPA explain, at least partially, the statistically (p < 0.05) longer bond lifetime for isolates associated with an infected cardiovascular device. PMID:22219202

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  18. 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.

  19. 1H, 13C and 15N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    PubMed

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

    2016-04-01

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

  20. 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.

  1. 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.

  2. (1)JCH NMR Profile: Identification of Key Structural Features and Functionalities by Visual Observation and Direct Measurement of One-Bond Proton-Carbon Coupling Constants.

    PubMed

    Marcó, Núria; Souza, Alexandre A; Nolis, Pau; Cobas, Carlos; Gil, Roberto R; Parella, Teodor

    2017-02-17

    A user-friendly NMR interface for the visual and accurate determination of experimental one-bond proton-carbon coupling constants ((1)JCH) in small molecules is presented. This intuitive (1)JCH profile correlates directly to δ((1)H), and (1)JCH facilitates the rapid identification and assignment of (1)H signals belonging to key structural elements and functional groups. Illustrative examples are provided for some target molecules, including terminal alkynes, strained rings, electronegative substituents, or lone-pair-bearing heteronuclei.

  3. Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

    PubMed

    Rout, Ashok K; Patel, Sunita; Somlata; Shukla, Manish; Saraswathi, Deepa; Bhattacharya, Alok; Chary, Kandala V R

    2013-08-09

    EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin.

  4. NMR structure reveals intramolecular regulation mechanism for pheromone binding and release

    PubMed Central

    Horst, Reto; Damberger, Fred; Luginbühl, Peter; Güntert, Peter; Peng, Guihong; Nikonova, Larisa; Leal, Walter S.; Wüthrich, Kurt

    2001-01-01

    Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBPA present at pH 4.5 and BmPBPB present at pH 6.5. Here, we describe the NMR structure of BmPBPA, which consists of a tightly packed arrangement of seven α-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four α-helices that forms the ligand binding site in the crystal structure of a BmPBP–pheromone complex is preserved in BmPBPA. The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, α7, which is located in the pheromone-binding site in the core of the unliganded BmPBPA. Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of α7 triggering the release of the pheromone from BmPBP to the membrane-standing receptor. PMID:11724947

  5. Substituent effects on axle binding in amide pseudorotaxanes: comparison of NMR titration and ITC data with DFT calculations.

    PubMed

    Kaufmann, Lena; Dzyuba, Egor V; Malberg, Friedrich; Löw, Nora L; Groschke, Matthias; Brusilowskij, Boris; Huuskonen, Juhani; Rissanen, Kari; Kirchner, Barbara; Schalley, Christoph A

    2012-08-14

    The binding behaviour of differently substituted diamide axle molecules to Hunter/Vögtle tetralactam macrocycles was studied with a combination of NMR titration, isothermal titration calorimetry (ITC) experiments and calculations employing density functional theory (DFT), along with dispersion-corrected exchange-correlation functionals. Guests with alkyl or alkenyl chains attached to the diamide carbonyl groups have a significantly higher binding affinity to the macrocycle than guests with benzoyl amides and their substituted analogues. While the binding of the benzoyl and alkenyl substituted axles is enthalpically driven, the alkyl-substituted guest binds mainly because of a positive binding entropy. The electronic effects of para-substituents at the benzoyl moieties have an influence on the binding affinities. Electron donating substituents increase, while electron-withdrawing substituents decrease the binding energies. The binding affinities obtained from both NMR titration and ITC experiments correlate well with each other. The substituent effects observed in the experimental data are reflected in adiabatic interaction energies calculated with density functional methods. The calculated structures also agree well with pseudorotaxane crystal structures.

  6. Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR.

    PubMed

    Assadi-Porter, Fariba M; Tonelli, Marco; Maillet, Emeline; Hallenga, Klaas; Benard, Outhiriaradjou; Max, Marianna; Markley, John L

    2008-06-11

    We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.

  7. Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy.

    PubMed

    Ragavan, Mukundan; Iconaru, Luigi I; Park, Cheon-Gil; Kriwacki, Richard W; Hilty, Christian

    2017-06-12

    The kinase inhibitory domain of the cell cycle regulatory protein p27(Kip1) (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of (13) C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Protein conformational changes studied by diffusion NMR spectroscopy: Application to helix-loop-helix calcium binding proteins

    PubMed Central

    Weljie, Aalim M.; Yamniuk, Aaron P.; Yoshino, Hidenori; Izumi, Yoshinobu; Vogel, Hans J.

    2003-01-01

    Pulsed-field gradient (PFG) diffusion NMR spectroscopy studies were conducted with several helix-loop-helix regulatory Ca2+-binding proteins to characterize the conformational changes associated with Ca2+-saturation and/or binding targets. The calmodulin (CaM) system was used as a basis for evaluation, with similar hydrodynamic radii (Rh) obtained for apo- and Ca2+-CaM, consistent with previously reported Rh data. In addition, conformational changes associated with CaM binding to target peptides from myosin light chain kinase (MLCK), phosphodiesterase (PDE), and simian immunodeficiency virus (SIV) were accurately determined compared with small-angle X-ray scattering results. Both sets of data demonstrate the well-established collapse of the extended Ca2+-CaM molecule into a globular complex upon peptide binding. The Rh of CaM complexes with target peptides from CaM-dependent protein kinase I (CaMKI) and an N-terminal portion of the SIV peptide (SIV-N), as well as the anticancer drug cisplatin were also determined. The CaMKI complex demonstrates a collapse analogous to that observed for MLCK, PDE, and SIV, while the SIV-N shows only a partial collapse. Interestingly, the covalent CaM–cisplatin complex shows a near complete collapse, not expected from previous studies. The method was extended to related calcium binding proteins to show that the Rh of calcium and integrin binding protein (CIB), calbrain, and the calcium-binding region from soybean calcium-dependent protein kinase (CDPK) decrease on Ca2+-binding to various extents. Heteronuclear NMR spectroscopy suggests that for CIB and calbrain this is likely because of shifting the equilibrium from unfolded to folded conformations, with calbrain forming a dimer structure. These results demonstrate the utility of PFG-diffusion NMR to rapidly and accurately screen for molecular size changes on protein–ligand and protein–protein interactions for this class of proteins. PMID:12538886

  9. Binding constant determination of high-affinity protein-ligand complexes by electrospray ionization mass spectrometry and ligand competition.

    PubMed

    Wortmann, Arno; Jecklin, Matthias C; Touboul, David; Badertscher, Martin; Zenobi, Renato

    2008-05-01

    We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.

  10. Protocols Utilizing Constant pH Molecular Dynamics to Compute pH-Dependent Binding Free Energies

    PubMed Central

    2015-01-01

    In protein–ligand binding, the electrostatic environments of the two binding partners may vary significantly in bound and unbound states, which may lead to protonation changes upon binding. In cases where ligand binding results in a net uptake or release of protons, the free energy of binding is pH-dependent. Nevertheless, conventional free energy calculations and molecular docking protocols typically do not rigorously account for changes in protonation that may occur upon ligand binding. To address these shortcomings, we present a simple methodology based on Wyman’s binding polynomial formalism to account for the pH dependence of binding free energies and demonstrate its use on cucurbit[7]uril (CB[7]) host–guest systems. Using constant pH molecular dynamics and a reference binding free energy that is taken either from experiment or from thermodynamic integration computations, the pH-dependent binding free energy is determined. This computational protocol accurately captures the large pKa shifts observed experimentally upon CB[7]:guest association and reproduces experimental binding free energies at different levels of pH. We show that incorrect assignment of fixed protonation states in free energy computations can give errors of >2 kcal/mol in these host–guest systems. Use of the methods presented here avoids such errors, thus suggesting their utility in computing proton-linked binding free energies for protein–ligand complexes. PMID:25134690

  11. 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

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

    Treesearch

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

    1997-01-01

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

  13. Probing the transient dark state of substrate binding to GroEL by relaxation-based solution NMR.

    PubMed

    Libich, David S; Fawzi, Nicolas L; Ying, Jinfa; Clore, G Marius

    2013-07-09

    The mechanism whereby the prototypical chaperonin GroEL performs work on substrate proteins has not yet been fully elucidated, hindered by lack of detailed structural and dynamic information on the bound substrate. Previous investigations have produced conflicting reports on the state of GroEL-bound polypeptides, largely due to the transient and dynamic nature of these complexes. Here, we present a unique approach, based on combined analysis of four complementary relaxation-based NMR experiments, to probe directly the "dark" NMR-invisible state of the model, intrinsically disordered, polypeptide amyloid β (Aβ40) bound to GroEL. The four NMR experiments, lifetime line-broadening, dark-state exchange saturation transfer, relaxation dispersion, and small exchange-induced chemical shifts, are dependent in different ways on the overall exchange rates and populations of the free and bound states of the substrate, as well as on residue-specific dynamics and structure within the bound state as reported by transverse magnetization relaxation rates and backbone chemical shifts, respectively. Global fitting of all the NMR data shows that the complex is transient with a lifetime of <1 ms, that binding involves two predominantly hydrophobic segments corresponding to predicted GroEL consensus binding sequences, and that the structure of the bound polypeptide remains intrinsically and dynamically disordered with minimal changes in secondary structure propensity relative to the free state. Our results establish a unique method to observe NMR-invisible dynamic states of GroEL-bound substrates and to describe at atomic resolution the events between substrate binding and encapsulation that are crucial for understanding the normal and stress-related metabolic function of chaperonins.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-24

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

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

    PubMed

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

    2011-08-05

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

  17. Determination of stability constants for thallium(III) cyanide complexes in aqueous solution by means of sup 13 C and sup 205 Ti NMR

    SciTech Connect

    Blixt, J.; Gyori, B.; Glaser, J. )

    1989-11-27

    In contrast to what is usually assumed, we have found and proved that thallium(III) forms very strong cyanide complexes in aqueous solution. The authors have investigated this system using {sup 205}Tl, {sup 13}C, and {sup 14}N NMR and potentiometry and established the existence of four thallium(III)-cyanide complexes of the composition Tl(CN){sub n}{sup 3-n}, n = 1-4. We have measured their chemical shifts and spin-spin coupling constants and determined their formation constants at 25{degree}C in dilute (0.05 M) aqueous solution in the ionic medium ((Na{sup +}) = 1 M, (Li{sup +}) + (H{sup +}) = 3 M, (ClO{sub 4}{sup {minus}}) = 4 M). We have also determined the stability constant for HCN in the same ionic medium, log K{sub a} = 10.11 (5).

  18. 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.

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

    PubMed

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

    1992-02-17

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

  20. 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.

  1. 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

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

    SciTech Connect

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

    2016-06-15

    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 ({sup 19}F and {sup 81}Br nuclei) measurements. We present results for 12, 12–ionenes in the presence of different halide ions (F{sup −}, Cl{sup −}, Br{sup −} and I{sup −}), 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.

  3. Investigations into the binding of jadomycin DS to human topoisomerase IIβ by WaterLOGSY NMR spectroscopy.

    PubMed

    Martinez-Farina, Camilo F; McCormick, Nicole; Robertson, Andrew W; Clement, Helen; Jee, Alison; Ampaw, Anna; Chan, Nei-Li; Syvitski, Ray T; Jakeman, David L

    2015-11-07

    The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIβ and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.

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

    PubMed

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

    2017-04-28

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

  5. Characterization of binding between 17β-estradiol and estriol with humic acid via NMR and biochemical analysis.

    PubMed

    Bedard, Mary; Giffear, Kelly A; Ponton, Lisa; Sienerth, Karl D; Del Gaizo Moore, Victoria

    2014-05-01

    Endocrine disruptors, such as 17β-estradiol (E2) and estriol (E3), are ubiquitously found in the environment and their presence has gained recognition as a significant health risk. Sorption of estrogens by dissolved organic matter (DOM) influences their concentration and effects. Although there is some experimental evidence suggesting that sorption occurs through hydrophobic interactions, there is not a complete understanding of this association. Therefore, we sought to more extensively characterize binding between humic acid (HA), a major component of DOM, with E2 and E3 using nuclear magnetic resonance spectroscopy (NMR). Our results indicate that binding between these estrogens and HA occurs primarily at the aromatic ring, and takes place even at relatively low HA concentration. Specific interactions between E2 and HA were confirmed by assessing binding via ELISA assay, and the effect of binding on estrogenic activity was studied via a yeast bioassay. Together, these studies build upon previously theorized interactions, yielding a more comprehensive model of binding between estrogens and HA, as well as demonstrate the value of the confluence of biochemical assay methods with analytical NMR techniques. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. 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

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

    PubMed

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

    2013-01-14

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

  8. NMR studies on the binding of antitumor drug nogalamycin to DNA hexamer d(CGTACG).

    PubMed

    Robinson, H; Liaw, Y C; van der Marel, G A; van Boom, J H; Wang, A H

    1990-08-25

    The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.

  9. 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

  10. 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.

  11. NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

    PubMed Central

    Fenwick, Michael K.; Oswald, Robert E.

    2008-01-01

    Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

  12. 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. © 2013 John Wiley & Sons A/S.

  13. Development of a dual cell, flow-injection sample holder, and NMR probe for comparative ligand-binding studies

    NASA Astrophysics Data System (ADS)

    Marquardsen, Thorsten; Hofmann, Martin; Hollander, Johan G.; Loch, Caroline M. P.; Kiihne, Suzanne R.; Engelke, Frank; Siegal, Gregg

    2006-09-01

    NMR based ligand screening is becoming increasingly important for the very early stages of drug discovery. We have proposed a method that makes highly efficient use of a single sample of a scarce target, or one with poor or limited solubility, to screen an entire compound library. This comparative method is based on immobilizing the target for the screening procedure. In order to support the method, a dual cell, flow injection probe with a single receiver coil has been constructed. The flow injection probe has been mated to a single high performance pump and sample handling system to enable the automated analysis of large numbers of compound mixes for binding to the target. The probe, having an 8 mm 1H/ 2H dual tuned coil and triple axis gradients, is easily shimmed and yields NMR spectra of comparable quality to a standard 5 mm high-resolution probe. The lineshape in the presence of a solid support is identical to that in glass NMR tubes in a 5 mm probe. Control spectra of each cell are identical and well separated, while ligand binding in a complex mixture can be readily detected in 20-30 min, thus paving the way for use of the probe for actual drug discovery efforts.

  14. Solid state {sup 31}P NMR study of phosphonate binding sites in guanidine-functionalized, molecular imprinted silica xerogels

    SciTech Connect

    Sasaki, D.Y.; Alam, T.D.

    2000-01-03

    Phosphonate binding sites in guanidine and ammonium surface-functionalized silica xerogels were prepared via the molecular imprinting technique and characterized using solid state {sup 31}P MAS NMR. One-point, two-point, and non-specific host-guest interactions between phenylphosphonic acid (PPA) and the functionalized gels were distinguished by characteristic chemical shifts of the observed absorption peaks. Using solid state as well as solution phase NMR analyses, absorptions observed at 15.5 ppm and 6.5 ppm were identified as resulting from the 1:1 (one-point) and 2:1 (two-point) guanidine to phosphonate interactions, respectively. Similar absorptions were observed with the ammonium functionalized gels. By examining the host-guest interactions within the gels, the efficiency of the molecular imprinting procedure with regard to the functional monomer-to-template interaction could be readily assessed. Template removal followed by substrate adsorption studies conducted on the guanidine functionalized gels provided a method to evaluate the binding characteristics of the receptor sites to a phosphonate substrate. During these experiments, {sup 29}Si and {sup 31}P MAS NMR acted as diagnostic monitors to identify structural changes occurring in the gel matrix and at the receptor site from solvent mediated processes.

  15. 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.

  16. Relationship between NMR shielding and heme binding strength for a series of 7-substituted quinolines.

    PubMed

    Casabianca, Leah B; de Dios, Angel C

    2006-06-29

    Chemical shielding tensors are calculated for the carbons in a series of 4-aminoquinolines with different substituents at the 7-position. The sigma(11) component is used as a measure of the relative pi-electron density at each carbon. By comparing the pi-electron density at each carbon with the log K of binding to heme (Kaschula et al. J. Med. Chem. 2002, 45, 3531), the drug-heme association is found to increase with increasing pi-electron density at the carbons meta to the substituent and with decreasing pi-electron density at the carbons ortho and para to the substituent. The greatest change in pi-electron density is at the ortho carbons, and log K increases with a decrease in pi-electron density on the ring containing the substituent, which corresponds to an increase in the pi-dipole between the two rings. An examination of the solution structures of the pi-pi complexes formed by amodiaquine and quinine with heme (Leed et al. Biochemistry 2002, 41, 10245. de Dios et al. Inorg. Chem. 2004, 43, 8078) shows that the pi-dipoles in each drug and in the porphyrin ring of heme may be paired. The chloro-substituted compound has an association constant that is an order of magnitude higher than the other compounds in the series, but the pi-electron density at the ring containing the substituent is not correspondingly low. This lack of correlation indicates that the Cl-substituted compound may be binding to heme in a manner that differs from the other compounds in the series.

  17. Constraining binding hot spots: NMR and MD simulations provide a structural explanation for enthalpy-entropy compensation in SH2-ligand binding

    PubMed Central

    Ward, Joshua M.; Gorenstein, Nina M.; Tian, Jianhua; Martin, Stephen F.; Post, Carol Beth

    2010-01-01

    NMR spectroscopy and molecular dynamics (MD) simulations were used to probe the structure and dynamics of complexes of three phosphotyrosine-derived peptides with the Src SH2 domain in an effort to uncover a structural explanation for enthalpy-entropy compensation observed in the binding thermodynamics. The series of phosphotyrosine peptide derivatives comprises the natural pYEEI Src SH2 ligand, a constrained mimic, in which the phosphotyrosine (pY) residue is preorganized in the bound conformation for the purpose of gaining an entropic advantage to binding, and a flexible analog of the constrained mimic. The expected gain in binding entropy of the constrained mimic was realized; however, a balancing loss in binding enthalpy was also observed that could not be rationalized from the crystallographic structures. We examined protein dynamics to evaluate whether the observed enthalpic penalty might be the result of effects arising from altered motions in the complex. 15N-relaxation studies and positional fluctuations from molecular dynamics indicate that the main-chain dynamics of the protein show little variation among the three complexes. Root mean squared (RMS) coordinate deviations vary by less than 1.5 Å for all non-hydrogen atoms for the crystal structures and in the ensemble average structures calculated from the simulations. In contrast to this striking similarity in the structures and dynamics, there are a number of large chemical shift differences from residues across the binding interface, but particularly from key Src SH2 residues that interact with pY, the ‘hot spot’ residue, which contributes about half of the binding free energy. Rank order correlations between chemical shifts and ligand binding enthalpy for several pY-binding residues, coupled with available mutagenesis and calorimetric data, suggest that subtle structural perturbations (< 1 Å) from the conformational constraint of the pY residue sufficiently alter the geometry of enthalpically

  18. Constraining binding hot spots: NMR and molecular dynamics simulations provide a structural explanation for enthalpy-entropy compensation in SH2-ligand binding.

    PubMed

    Ward, Joshua M; Gorenstein, Nina M; Tian, Jianhua; Martin, Stephen F; Post, Carol Beth

    2010-08-18

    NMR spectroscopy and molecular dynamics (MD) simulations were used to probe the structure and dynamics of complexes of three phosphotyrosine-derived peptides with the Src SH2 domain in an effort to uncover a structural explanation for enthalpy-entropy compensation observed in the binding thermodynamics. The series of phosphotyrosine peptide derivatives comprises the natural pYEEI Src SH2 ligand, a constrained mimic, in which the phosphotyrosine (pY) residue is preorganized in the bound conformation for the purpose of gaining an entropic advantage to binding, and a flexible analogue of the constrained mimic. The expected gain in binding entropy of the constrained mimic was realized; however, a balancing loss in binding enthalpy was also observed that could not be rationalized from the crystallographic structures. We examined protein dynamics to evaluate whether the observed enthalpic penalty might be the result of effects arising from altered motions in the complex. (15)N-relaxation studies and positional fluctuations from molecular dynamics indicate that the main-chain dynamics of the protein show little variation among the three complexes. Root mean squared (rms) coordinate deviations vary by less than 1.5 A for all non-hydrogen atoms for the crystal structures and in the ensemble average structures calculated from the simulations. In contrast to this striking similarity in the structures and dynamics, there are a number of large chemical shift differences from residues across the binding interface, but particularly from key Src SH2 residues that interact with pY, the "hot spot" residue, which contributes about one-half of the binding free energy. Rank-order correlations between chemical shifts and ligand binding enthalpy for several pY-binding residues, coupled with available mutagenesis and calorimetric data, suggest that subtle structural perturbations (<1 A) from the conformational constraint of the pY residue sufficiently alter the geometry of enthalpically

  19. 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.

  20. NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine.

    PubMed

    Duchardt-Ferner, Elke; Juen, Michael; Kreutz, Christoph; Wöhnert, Jens

    2017-04-01

    RNA aptamers are used in a wide range of biotechnological or biomedical applications. In many cases the high resolution structures of these aptamers in their ligand-complexes have revealed fundamental aspects of RNA folding and RNA small molecule interactions. Fluorescent RNA-ligand complexes in particular find applications as optical sensors or as endogenous fluorescent tags for RNA tracking in vivo. Structures of RNA aptamers and aptamer ligand complexes constitute the starting point for rational function directed optimization approaches. Here, we present the NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine (TMR) in complex with the ligand 5-carboxy-tetramethylrhodamine (5-TAMRA) as a starting point for a high-resolution structure determination using NMR spectroscopy in solution.

  1. Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A.

    PubMed

    Kawamura, Takahiro; Wakamoto, Takuro; Kitazawa, Soichiro; Sakuraba, Shun; Kameda, Tomoshi; Kitahara, Ryo

    2017-05-09

    Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r(6) distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed

    Sever, Mary J; Wilker, Jonathan J

    2006-02-14

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

  3. Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen clusters.

    PubMed

    Matsumoto-Takasaki, Ayano; Hanashima, Shinya; Aoki, Ami; Yuasa, Noriyuki; Ogawa, Haruhiko; Sato, Reiko; Kawakami, Hiroko; Mizuno, Mamoru; Nakada, Hiroshi; Yamaguchi, Yoshiki; Fujita-Yamaguchi, Yoko

    2012-03-01

    Tn-antigens are tumour-associated carbohydrate antigens that are involved in metastatic processes and are associated with a poor prognosis. MLS128 monoclonal antibody recognizes the structures of two or three consecutive Tn-antigens (Tn2 or Tn3). Since MLS128 treatment inhibits colon and breast cancer cell growth [Morita, N., Yajima, Y., Asanuma, H., Nakada, H., and Fujita-Yamaguchi, Y. (2009) Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Biosci. Trends 3, 32-37.], understanding the interaction between MLS128 and Tn-clusters may allow us to the development of novel cancer therapeutics. Although MLS128 was previously reported to have specificity for Tn3 rather than Tn2, similar levels of Tn2/Tn3 binding were unexpectedly observed at 37°C. Thus, thermodynamic analyses were performed via surface plasmon resonance (SPR) using synthetic Tn2- and Tn3-peptides at 10, 15, 20, 25 and 30°C. SPR results revealed that MLS128's association constants for both antigens were highly temperature dependent. Below 25°C MLS128's association constant for Tn3-peptide was clearly higher than that for Tn2-peptide. At 30°C, however, the association constant for Tn2-peptide was higher than that for Tn3-peptide. This reversal of affinity is due to the sharp increase in K(d) for Tn3. These results were confirmed by NMR, which directly measured MLS128-Tn binding in solution. This study suggested that thermodynamic control plays a critical role in the interaction between MLS128/Tn2 and MLS128/Tn3.

  4. 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.

  5. Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5.

    PubMed

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Cabana, Jérôme; Lavigne, Pierre; LeHoux, Jean-Guy

    2013-11-01

    STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

  6. Ligand-Detected Relaxation Dispersion NMR Spectroscopy: Dynamics of preQ1–RNA Binding**

    PubMed Central

    Moschen, Thomas; Wunderlich, Christoph Hermann; Spitzer, Romana; Levic, Jasmin; Micura, Ronald; Tollinger, Martin; Kreutz, Christoph

    2015-01-01

    An NMR-based approach to characterizing the binding kinetics of ligand molecules to biomolecules, like RNA or proteins, by ligand-detected Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments is described. A 15N-modified preQ1 ligand is used to acquire relaxation dispersion experiments in the presence of low amounts of the Fsu class I preQ1 aptamer RNA, and increasing ligand concentrations to probe the RNA small molecule interaction. Our experimental data strongly support the conformational selection mechanism postulated. The approach gives direct access to two parameters of a ligand–receptor interaction: the off rate and the population of the small molecule–receptor complex. A detailed description of the kinetics underlying the ligand binding process is of crucial importance to fully understanding a riboswitch’s function and to evaluate potential new antibiotics candidates targeting the noncoding RNA species. Ligand-detected NMR relaxation dispersion experiments represent a valuable diagnostic tool for the characterization of binding mechanisms. PMID:25403518

  7. Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

    PubMed Central

    Kinde, Monica N.; Bondarenko, Vasyl; Granata, Daniele; Bu, Weiming; Grasty, Kimberly C.; Loll, Patrick J.; Carnevale, Vincenzo; Klein, Michael L.; Eckenhoff, Roderic G.; Tang, Pei

    2016-01-01

    Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4–S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4–S5 linker and at a critical hinge that controls the gating and inactivation motion of S6. PMID:27856739

  8. 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.

  9. 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

    2016-07-06

    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.

  10. Binding constants determined from Ca2+ current responses to rapid applications and washouts of nifedipine in frog cardiac myocytes.

    PubMed

    Méry, P F; Hove-Madsen, L; Mazet, J L; Hanf, R; Fischmeister, R

    1996-07-01

    1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a

  11. NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

    PubMed

    Espinosa, J F; Asensio, J L; García, J L; Laynez, J; Bruix, M; Wright, C; Siebert, H C; Gabius, H J; Cañada, F J; Jiménez-Barbero, J

    2000-07-01

    The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.

  12. Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR.

    PubMed

    Roth, Günter; Freund, Stefan; Möhrle, Bernd; Wöllner, Karin; Brünjes, Jente; Gauglitz, Günter; Wiesmüller, Karl-Heinz; Jung, Günther

    2007-02-12

    Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH(2) as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 muM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. (1)H,(15)N heteronuclear NMR localised the interaction site across the beta sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.

  13. 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.

  14. Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

    PubMed

    Acuña Castroviejo, D; del Aguila, C M; Fernández, B; Gomar, M D; Castillo, J L

    1991-06-24

    The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.

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

    PubMed

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

    2011-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

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

    PubMed

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

    2015-11-01

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

  18. Tb3+ and Ca2+ binding to phosphatidylcholine. A study comparing data from optical, NMR, and infrared spectroscopies.

    PubMed Central

    Petersheim, M; Halladay, H N; Blodnieks, J

    1989-01-01

    The paramagnetic and luminescent lanthanides are unique probes of cation-phospholipid interactions. Their spectroscopic properties provide the means to characterize and monitor complexes formed with lipids in ways not possible with biochemically more interesting cations, such as Ca2+. In this work, Tb3+-phosphatidylcholine complexes are described using the luminescence properties of Tb3+, the effect of its paramagnetism on the 31P NMR and 13C NMR spectra of the lipid, and changes in the infrared spectrum of the lipid induced by the cation. There are two Tb3+-phosphatidylcholine complexes with very different coordination environments, as evidenced by changes in the optical excitation spectrum of the lanthanide. The NMR experiments indicate that the two complexes differ in the number of phosphate groups directly coordinating Tb3+. Tb3+ binding induces changes in the phosphodiester infrared bands that are most consistent with bidentate chelation of Tb3+ by each phosphate, whereas Ca2+-induced changes are more consistent with monodentate coordination. The significance of this discrepancy is discussed. PMID:2790138

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

    PubMed Central

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

    2009-01-01

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

  20. 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…

  1. 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…

  2. Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR

    PubMed Central

    Vetticatt, Mathew J.; Itin, Boris; Evans, Gary B.; Schramm, Vern L.

    2013-01-01

    Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4′-deaza-1′-aza-2′-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to “achieve” the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state. PMID:24043827

  3. 13C-detected NMR experiments for measuring chemical shifts and coupling constants in nucleic acid bases.

    PubMed

    Fiala, Radovan; Sklenár, Vladimír

    2007-10-01

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

  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. Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

    PubMed Central

    Nissim, A; Jouvin, M H; Eshhar, Z

    1991-01-01

    Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI. Images PMID:1824934

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

    PubMed Central

    Meneses, Erick; Mittermaier, Anthony

    2014-01-01

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

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

    PubMed

    Meneses, Erick; Mittermaier, Anthony

    2014-10-03

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

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

    PubMed

    Cevc, Gregor

    2015-05-01

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

  9. Design of a hyperpolarized (15)N NMR probe that induces a large chemical-shift change upon binding of calcium ions.

    PubMed

    Hata, Ryunosuke; Nonaka, Hiroshi; Takakusagi, Yoichi; Ichikawa, Kazuhiro; Sando, Shinsuke

    2015-08-07

    Ca(2+) is a fundamental metal ion for physiological functioning. Therefore, molecular probes for Ca(2+) analysis are required. Recently, a hyperpolarized NMR probe has emerged as a promising tool. Here, we report a new design of a hyperpolarized NMR probe for Ca(2+), which showed a large chemical shift change upon binding to Ca(2+) and was applied for Ca(2+) sensing in a hyperpolarized state.

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

    PubMed

    Kempgens, Pierre; Rosé, Jacky

    2011-03-01

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

  11. NMR solution structures of adducts derived from the binding of polycyclic aromatic diol epoxides to DNA

    SciTech Connect

    Cosman, M.; Patel, D.J.; Hingerty, B.E.; Amin, S.; Broyde, S.; Geacintov, N.E.

    1995-12-31

    Site-specifically modified oligonucleotides were derived from the reactions of stereoisomeric polycyclic aromatic diol epoxide metabolite model compounds with oligonucleotides of defined base composition and sequence. The NMR solution structures of ten different adducts studied so far are briefly described, and it is shown that stereochemical factors and the nature of the oligonucleotide context of the complementary strands, exert a powerful influence on the conformational features of these adducts.

  12. Constant domains influence binding of mouse–human chimeric antibodies to the capsular polypeptide of Bacillus anthracis

    PubMed Central

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-01-01

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct “rim” quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse–human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared “puffy” in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site. PMID:23863605

  13. Constant domains influence binding of mouse-human chimeric antibodies to the capsular polypeptide of Bacillus anthracis.

    PubMed

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-08-15

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct "rim" quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse-human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared "puffy" in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site.

  14. 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].

  15. 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

  16. 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)…

  17. 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)…

  18. NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae*

    PubMed Central

    Nedielkov, Ruslan; Steffen, Wojtek; Steuber, Julia; Möller, Heiko M.

    2013-01-01

    The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR. PMID:24003222

  19. On the Usage of Locally Dense Basis Sets in the Calculation of NMR Indirect Nuclear Spin-Spin Coupling Constants

    NASA Astrophysics Data System (ADS)

    Sanchez, Marina; Provasi, Patricio F.; Aucar, Gustavo A.; Sauer, Stephan P. A.

    Locally dense basis sets (constants shows that the non-contact orbital paramagnetic term is the most important contribution followed by the also non-contact spin-dipolar term. The Fermi contact term is the largest contribution only in the synperiplanar conformations of 1,2-difluoroethane and -propane.

  20. Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

    SciTech Connect

    Brito, R.M.M.; Putkey, J.A.; Rosevear, P.R. ); Strynadka, N.C.J.; James, M.N.G. )

    1991-10-22

    One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca{sup 2+} binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the {sup 1}H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. The authors have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca{sup 2+} at site II. Comparison of the apo, Mg{sup 2+}-, and Ca{sup 2+}-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but it is consequence of the inability of CBM-IIA to bind CA{sup 2+} at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed.

  1. 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.

  2. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein.

    PubMed Central

    DeJong, Eric S; Marzluff, William F; Nikonowicz, Edward P

    2002-01-01

    The 3' end of replication-dependent histone mRNAs terminate in a conserved sequence containing a stem-loop. This 26-nt sequence is the binding site for a protein, stem-loop binding protein (SLBP), that is involved in multiple aspects of histone mRNA metabolism and regulation. We have determined the structure of the 26-nt sequence by multidimensional NMR spectroscopy. There is a 16-nt stem-loop motif, with a conserved 6-bp stem and a 4-nt loop. The loop is closed by a conserved U.A base pair that terminates the canonical A-form stem. The pyrimidine-rich 4-nt loop, UUUC, is well organized with the three uridines stacking on the helix, and the fourth base extending across the major groove into the solvent. The flanking nucleotides at the base of the hairpin stem do not assume a unique conformation, despite the fact that the 5' flanking nucleotides are a critical component of the SLBP binding site. PMID:11871662

  4. Calculation of equilibrium binding constants and cooperativity of Cu(II) mixed solvated complexes formation.

    PubMed

    Kudrev, A G

    2012-11-15

    A new extension of matrix approach is proposed to calculate the equilibrium constants of coordinated solvent substitution in a metal ion first salvation shell in the mixed solvent system. The proposed method allows reducing the number of independent variables, necessary to calculate the fractions of species in solution. The equilibrium model of MeCN substitution with DMF and DMSO in the presence of Cu(II) ion for the assessment of structure of intermediate species is presented and verified. The distribution diagrams of Cu(II) species in mixed organic solvents have been analyzed using the modified matrix method. The intrinsic equilibrium constants K of the first solvent molecule replacement in the Cu(II) coordination shell and the correction for the mutual influence between the solvent molecules as ligands in the successive complex formation (cooperativity parameter w) in acetonitrile solution have been calculated from the fitting procedure. It is shown that anticooperative substitution of MeCN by donor ligands in the first coordination shell of the Cu(II) ion is always governed by the change of coordination number during the stepwise process.

  5. SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase

    PubMed Central

    Gorrini, Marina; Lupi, Anna; Iadarola, Paolo; Santos, Conceição Dos; Rognoni, Paola; Dalzoppo, Daniele; Carrabino, Natalia; Pozzi, Ernesto; Baritussio, Aldo; Luisetti, Maurizio

    2005-01-01

    Background α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction. Methods and results At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin. Conclusion We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents. PMID:16351724

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

    PubMed

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

    2015-10-28

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

  7. 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.

  8. 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.

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

    SciTech Connect

    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 {sup 1}H/{sup 13}C/{sup 1}H chemical shift correlation spectrum by incorporating an additional cross-polarization period in the CTUC-COSY pulse sequence to enable proton chemical shift evolution and proton detection in the incrementable t{sub 1} and t{sub 3} periods, respectively. In addition to through-space and through-bond {sup 13}C/{sup 1}H and {sup 13}C/{sup 13}C chemical shift correlations, the 3D {sup 1}H/{sup 13}C/{sup 1}H experiment also provides a COSY-type {sup 1}H/{sup 1}H chemical shift correlation spectrum, where only the chemical shifts of those protons, which are bonded to two neighboring carbons, are correlated. By extracting 2D F1/F3 slices ({sup 1}H/{sup 1}H chemical shift correlation spectrum) at different {sup 13}C chemical shift frequencies from the 3D {sup 1}H/{sup 13}C/{sup 1}H spectrum, resonances of proton atoms located close to a specific carbon atom can be identified. Overall, the through-bond and through-space homonuclear/heteronuclear proximities determined from the

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

    PubMed Central

    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

  11. 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.

  12. NMR assignment and secondary structure of coiled coil domain of C-terminal myosin binding subunit of myosin phosphatase.

    PubMed

    Sharma, Alok K; Rigby, Alan C

    2014-07-01

    Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

  13. 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

    2012-03-08

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

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

    NASA Astrophysics Data System (ADS)

    Sahu, Sivabrata; Parashar, S. K. S.; Rout, G. C.

    2017-07-01

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

  15. A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding.

    PubMed

    Modi, S; Paine, M J; Sutcliffe, M J; Lian, L Y; Primrose, W U; Wolf, C R; Roberts, G C

    1996-04-09

    The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.

  16. Triosephosphate isomerase: 15N and 13C chemical shift assignments and conformational change upon ligand binding by magic-angle spinning solid-state NMR spectroscopy.

    PubMed

    Xu, Yimin; Lorieau, Justin; McDermott, Ann E

    2010-03-19

    Microcrystalline uniformly (13)C,(15)N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.

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

    PubMed

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

    2017-06-01

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

  18. 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

  19. 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

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

    PubMed

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

    2016-11-23

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

  1. 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...

  2. 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...

  3. The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

    PubMed

    Zamora-Carreras, Héctor; Torres, María; Bustamante, Noemí; Macedo, Anjos L; Rodríguez, Rosalía; Villalba, Mayte; Bruix, Marta

    2015-08-15

    Ole e 9 and Fra e 9 are two allergenic β-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

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

    PubMed

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

    2005-11-01

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

  5. Determination of drug-polymer binding constants by affinity capillary electrophoresis for aryl propionic acid derivatives and related compounds.

    PubMed

    Jia, Zhongjiang; Choi, Duk Soon; Chokshi, Hitesh

    2013-03-01

    The binding constants (K(b)s) of 17 aryl propionic acid derivatives (APADs) and related compounds with polyvinylpyrrolidone (PVP K30) and vinylpyrrolidone-vinyl acetate copolymer (Kollidon VA64) in aqueous media were determined by affinity capillary electrophoreses (ACE). The K(b)s of APAD to polymers increase with octanol-water partition coefficients of the compounds. Kollidon VA64 is a stronger binder than PVP K30 to APAD compounds. The K(b)s are greater at pH 4 than at pH 9. Both hydrophobic interaction and hydrogen bonding may be involved. However, hydrophobic interaction appears to be dominant. The ACE method is simple and fast, which could be used to study drug-polymer interaction in aqueous media.

  6. Measurement of binding constants of poly(ethylenimine) with metal ions and metal chelates in aqueous media by ultrafiltration

    SciTech Connect

    Juang, R.S.; Chen, M.N.

    1996-06-01

    Equilibrium constants for the binding of poly(ethylenimine) (PEI) with metal ions and metal chelates of ethylenediaminetetraacetic acid and nitrilotriacetic acid in the aqueous sulfate solutions were determined by batch ultrafiltration (UF) in the pH range of 3.0--3.8. The average coordination number of PEI groups interacting with one metal ion and metal chelate was also obtained by varying the initial concentration ratio of PEI to metal ion. A simple chemical equilibrium model proposed in this work enabled the authors to satisfactorily predict the rejection coefficient of UF of metal ions and metal chelates in the presence of PEI. Also, the effect of the formation of soluble hydroxy complexes of metal chelates on their retention was emphasized.

  7. 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…

  8. 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…

  9. Expression, purification, and solid-state NMR characterization of the membrane binding heme protein nitrophorin 7 in two electronic spin states.

    PubMed

    Varghese, Sabu; Yang, Fei; Pacheco, Victor; Wrede, Kathrin; Medvedev, Alexander; Ogata, Hideaki; Knipp, Markus; Heise, Henrike

    2013-10-08

    The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.

  10. Simple and rapid quantification of serotonin transporter binding using [(11)C]DASB bolus plus constant infusion.

    PubMed

    Gryglewski, G; Rischka, L; Philippe, C; Hahn, A; James, G M; Klebermass, E; Hienert, M; Silberbauer, L; Vanicek, T; Kautzky, A; Berroterán-Infante, N; Nics, L; Traub-Weidinger, T; Mitterhauser, M; Wadsak, W; Hacker, M; Kasper, S; Lanzenberger, R

    2017-04-01

    In-vivo quantification of serotonin transporters (SERT) in human brain has been a mainstay of molecular imaging in the field of neuropsychiatric disorders and helped to explore the underpinnings of several medical conditions, therapeutic and environmental influences. The emergence of PET/MR hybrid systems and the heterogeneity of SERT binding call for the development of efficient methods making the investigation of larger or vulnerable populations with limited scanner time and simultaneous changes in molecular and functional measures possible. We propose [(11)C]DASB bolus plus constant infusion for these applications and validate it against standard analyses of dynamic PET data. [(11)C]DASB bolus/infusion optimization was performed on data acquired after [(11)C]DASB bolus in 8 healthy subjects. Subsequently, 16 subjects underwent one scan using [(11)C]DASB bolus plus constant infusion with Kbol 160-179min and one scan after [(11)C]DASB bolus for inter-method reliability analysis. Arterial blood sampling and metabolite analysis were performed for all scans. Distribution volumes (VT) were obtained using Logan plots for bolus scans and ratios between tissue and plasma parent activity for bolus plus infusion scans for different time spans of the scan (VT-70 for 60-70min after start of tracer infusion, VT-90 for 75-90min, VT-120 for 100-120min) in 9 subjects. Omitting blood data, binding potentials (BPND) obtained using multilinear reference tissue modeling (MRTM2) and cerebellar gray matter as reference region were compared in 11 subjects. A Kbol of 160min was observed to be optimal for rapid equilibration in thalamus and striatum. VT-70 showed good intraclass correlation coefficients (ICCs) of 0.61-0.70 for thalamus, striatal regions and olfactory cortex with bias ≤5.1% compared to bolus scans. ICCs increased to 0.72-0.78 for VT-90 and 0.77-0.93 for VT-120 in these regions. BPND-90 had negligible bias ≤2.5%, low variability ≤7.9% and ICCs of 0.74-0.87; BPND-120

  11. (19) F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-β-Lactamase.

    PubMed

    Abboud, Martine I; Hinchliffe, Philip; Brem, Jürgen; Macsics, Robert; Pfeffer, Inga; Makena, Anne; Umland, Klaus-Daniel; Rydzik, Anna M; Li, Guo-Bo; Spencer, James; Claridge, Timothy D W; Schofield, Christopher J

    2017-03-27

    Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe (19) F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-Zn(II) active site, were selectively (19) F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.

  12. A solution NMR study of the interactions of oligomannosides and the anti-HIV-1 2G12 antibody reveals distinct binding modes for branched ligands.

    PubMed

    Enríquez-Navas, Pedro M; Marradi, Marco; Padro, Daniel; Angulo, Jesús; Penadés, Soledad

    2011-02-01

    The structural and affinity details of the interactions of synthetic oligomannosides, linear (di-, tri-, and tetra-) and branched (penta- and hepta-), with the broadly neutralizing anti-HIV-1 antibody 2G12 (HIV=human immunodeficiency virus) have been investigated in solution by using ligand-based NMR techniques, specifically saturation transfer difference (STD) NMR spectroscopy and transferred NOE experiments. Linear oligomannosides show similar binding modes to the antibody, with the nonreducing terminal disaccharide Manα(1→2)Man (Man=mannose) making the closest protein/ligand contacts in the bound state. In contrast, the branched pentamannoside shows two alternate binding modes, involving both ligand arms (D2- and D3-like), a dual binding description of the molecular recognition of this ligand by 2G12 in solution that differs from the single binding mode deduced from X-ray studies. On the contrary, the antibody shows an unexpected selectivity for one arm (D1-like) of the other branched ligand (heptamannoside). This result explains the previously reported lack of affinity enhancement relative to that of the D1-like tetramannoside. Single-ligand STD NMR titration experiments revealed noticeable differences in binding affinities among the linear and branched ligands in solution, with the latter showing decreased affinity. Among the analyzed series of ligands, the strongest 2G12 binders were the linear tri- and tetramannosides because both show similar affinity for the antibody. These results demonstrate that NMR spectroscopic techniques can deliver abundant structural, dynamics, and affinity information for the characterization of oligomannose-2G12 binding in solution, thus complementing, and, as in the case of the pentamannoside, extending, the structural view from X-ray crystallography. This information is of key importance for the development of multivalent synthetic gp120 high-mannose glycoconjugate mimics in the context of vaccine development.

  13. Exchanging Murine and Human Immunoglobulin Constant Chains Affects the Kinetics and Thermodynamics of Antigen Binding and Chimeric Antibody Autoreactivity

    PubMed Central

    Torres, Marcela; Fernandez-Fuentes, Narcis; Fiser, András; Casadevall, Arturo

    2007-01-01

    Mouse-human chimeric antibodies composed of murine variable (V) and human (C) chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of CH glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb) 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch) and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by CH region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the CH domains through an allosteric effect involving networks of highly connected amino acids. PMID:18074033

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

    PubMed

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

    2009-12-31

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

  15. NMR Chemical Shielding and Spin-Spin Coupling Constants of Liquid NH3: A Systematic Investigation using the Sequential QM/MM Method

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  16. 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.

  17. Proton and tritium NMR relaxation studies of peptide inhibitor binding to bacterial collagenase: Conformation and dynamics

    SciTech Connect

    Dive, V.; Lai, A.; Valensin, G.; Saba, G.; Yiotakis, A.; Toma, F. )

    1991-02-15

    The interaction of succinyl-Pro-Ala, a competitive inhibitor of Achromobacter iophagus collagenase, with the enzyme was studied by longitudinal proton and tritium relaxation. Specific deuterium and tritium labeling of the succinyl part at vicinal positions allowed the measurement of the cross-relaxation rates of individual proton or tritium spin pairs in the inhibitor-enzyme complex as well as in the free inhibitor. Overall correlation times, internuclear distances, and qualitative information on the internal mobility in Suc1 (as provided by the generalized order parameter S2) could be deduced by the comparison of proton and tritium cross-relaxation of spin pairs at complementary positions in the -CH2- CH2- moiety as analyzed in terms of the model-free approach by Lipari and Szabo. The conformational and motional parameters of the inhibitor in the free and enzyme-bound state were directly compared by this method. The measurement of proton cross-relaxation in the Ala residue provided additional information on the inhibitor binding. The determination of the order parameter in different parts of the inhibitor molecule in the bound state indicates that the succinyl and alanyl residues are primarily involved in the interaction with the enzyme activity site. The succinyl moiety, characterized in solution by the conformational equilibrium among the three staggered rotamers--i.e., trans: 50%; g+: 20%; g-: 30%--adopted in the bound state the unique trans conformation.

  18. 1H NMR studies of the 5-(hydroxymethyl)-2'-deoxyuridine containing TF1 binding site.

    PubMed Central

    Pasternack, L B; Bramham, J; Mayol, L; Galeone, A; Jia, X; Kearns, D R

    1996-01-01

    The pyrimidine base 5-(hydroxymethyl)-2'-deoxyuridine (HmU) is a common nucleotide in SPO1 phage DNA. Numerous transcriptional proteins bind HmU-containing DNA preferentially implicating a regulatory function of HmU. We have investigated the conformation and dynamics of d-(5'-CHmUCHmUACACGHmUGHmUAGAG-OH-3')2 (HmU-DNA). This oligonucleotide mimics the consensus sequence of Transcription Factor 1 (TF1). The HmU-DNA was compared to the thymine-containing oligonucleotide. NOESY and DQF COSY spectroscopy provided resonance assignments of nonexchangeable and exchangeable protons, intranucleotide, internucleotide and intrastrand proton-proton distances, and dihedral angle constraints. Methylene protons of the hydroxymethyl group are nonequivalent protons and the hydroxymethyl group is not freely rotating. The hydroxymethyl group adopts a specific orientation with the OH group oriented on the 3' side of the plane of the base. Analysis of imino proton resonances and NOEs indicates additional end base pair fraying and a temperature-induced transition to a conformation in which the internal HmU-A base pairs are disrupted or have reduced lifetimes. Orientation of the hydroxymethyl group indicates the presence of internucleotide intrastrand hydrogen bonding between the HmU12C5 hydroxyl group and A13. All sugars in both DNAs show a C2'endo conformation (typical of B-DNA). PMID:8759005

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

    PubMed

    Okada, Kyle S; Lee, Youngsoo

    2017-07-01

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

  20. Domain structure of antithrombin III. Tentative localization of the heparin binding region using /sup 1/H NMR spectroscopy

    SciTech Connect

    Gettins, P.; Wooten, E.W.

    1987-07-14

    The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by /sup 1/H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.

  1. 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

    2012-05-08

    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.

  2. The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to (d(ACGTACGT)) sub 2 and (d(TCGATCGA)) sub 2

    SciTech Connect

    Gilbert, D.E.; Feigon, J. )

    1991-03-05

    The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers (d(ACGTACGT)){sub 2} and (d(TCGATCGA)){sub 2} have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic ad chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer (d(ACGTACGT)){sub 2} is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to (d(TCGATCGA)){sub 2} is not cooperative. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While the authors conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.

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

    PubMed

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

    2016-04-14

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

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

    PubMed

    Mittermaier, Anthony; Meneses, Erick

    2013-01-01

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

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

    PubMed

    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.

  6. 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.

  7. 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.

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

    NASA Astrophysics Data System (ADS)

    Jayalakshmi, V.; Rama Krishna, N.

    2004-05-01

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

  9. 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.

  10. 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.

  11. Calculating the response of NMR shielding tensor σ(31P) and 2J(31P,13C) coupling constants in nucleic acid phosphate to coordination of the Mg2+ cation.

    PubMed

    Benda, Ladislav; Schneider, Bohdan; Sychrovský, Vladimír

    2011-03-24

    Dependence of NMR (31)P shielding tensor and (2)J(P,C) coupling constants on solvation of nucleic acid phosphate by Mg(2+) and water was studied using methods of bioinformatic structural analyses of crystallographic data and DFT B3LYP calculations of NMR parameters. The effect of solvent dynamics on NMR parameters was calculated using molecular dynamic. The NMR calculations for representative solvation patterns determined in crystals of B-DNA and A-RNA molecules pointed out the crucial importance of local Mg(2+) coordination geometry, including hydration by explicit water molecules and necessity of dynamical averaging over the solvent reorientation. The dynamically averaged (31)P chemical shift decreased by 2-9.5 ppm upon Mg(2+) coordination, the chemical shielding anisotropy increased by 0-20 ppm, and the (2)J(P,C5') coupling magnitude decreased by 0.2-1.8 Hz upon Mg(2+) coordination. The calculated decrease of the (31)P chemical shift is in excellent agreement with the 1.5-10 ppm decrease of the phosphorothioate (31)P chemical shift upon Cd(2+) coordination probed experimentally in hammerhead ribozyme (Suzumura; et al. J. Am. Chem. Soc. 2002, 124, 8230-8236; Osborne; et al., Biochemistry 2009, 48, 10654-10664). None of the dynamically averaged NMR parameters unequivocally distinguishes the site-specific Mg(2+) coordination to one of the two nonesterified phosphate oxygen atoms of the phosphate determined by bioinformatic analyses. By comparing the limit cases of static and dynamically averaged solvation, we propose that mobility of the solvent has a dramatic impact on NMR parameters of nucleic acid phosphate and must be taken into account for their accurate modeling.

  12. 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

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

    SciTech Connect

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

    2016-03-17

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

  14. 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.

  15. Model-independent link between the macroscopic and microscopic descriptions of multidentate macromolecular binding: relationship between stepwise, intrinsic, and microscopic equilibrium constants.

    PubMed

    Lluís Garcés, Josep; Rey-Castro, Carlos; David, Calin; Madurga, Sergio; Mas, Francesc; Pastor, Isabel; Puy, Jaume

    2009-11-19

    The binding of ions or other small molecules to macromolecules and surfaces can be macroscopically characterized by means of the stepwise (or stoichiometric) equilibrium constants, which can be obtained experimentally from coverage versus concentration data. The present work presents a novel, simple, and direct interpretation of the stepwise constants in terms of the microscopic, site-specific, stability constants. This formalism can be applied to the most general case, including the heterogeneity of the sites, interactions among them, multicomponent adsorption, and so forth, and, in particular, to chelate complexation. We show that the stepwise equilibrium constants can be expressed as a product of two factors, (i) the average number of free potential sites (per bound ion) of the microscopic species to be complexed (stoichiometric factor) and (ii) the average of the microscopic stability constants of their free potential sites. The latter factor generalizes the concept of the intrinsic equilibrium constant to systems with chelate complexation and reduces to the standard definition for monodentate binding. However, in the case of heterogeneous multidentate complexation, the stoichiometric factor cannot be known a priori, so that the finding of the intrinsic constants is not trivial. One option is to approximate the stoichiometric factor by the value that would correspond to identical active centers. We investigate the accuracy of this assumption by comparing the resulting approximate intrinsic constants to those obtained by Monte Carlo simulation of several binding models. For the cases investigated, it is found that the assumption is quite accurate when no correlated structures (typical of short-range interactions) are formed along the chain. For adsorption of particles attached to a large number of active centers, the formalism presented here leads to the Widom particle insertion method.

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

    PubMed

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

    2017-04-15

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

  17. Accurate determination of pyridine-poly(amidoamine) dendrimer absolute binding constants with the OPLS-AA force field and direct integration of radial distribution functions.

    PubMed

    Peng, Yong; Kaminski, George A

    2005-08-11

    OPLS-AA force field and direct integration of intermolecular radial distribution functions (RDF) were employed to calculate absolute binding constants of pyridine molecules to amino group (NH2) and amide group hydrogen atoms in and first generation poly(amidoamine) dendrimers in chloroform. The average errors in the absolute and relative association constants, as predicted with the calculations, are 14.1% and 10.8%, respectively, which translate into ca. 0.08 and 0.06 kcal/mol errors in the absolute and relative binding free energies. We believe that this level of accuracy proves the applicability of the OPLS-AA, force field, in combination with the direct RDF integration, to reproducing and predicting absolute intermolecular association constants of low magnitudes (ca. 0.2-2.0 range).

  18. Accurate Determination of Pyridine -- Poly (Amidoamine) Dendrimer Absolute Binding Constants with the OPLS-AA Force Field and Direct Integration of Radial Distribution Functions

    NASA Astrophysics Data System (ADS)

    Peng, Yong; Kaminski, George

    2006-03-01

    OPLS-AA force field and direct integration of intermolecular radial distribution functions (RDF) were employed to calculate absolute binding constants of pyridine molecules to NH2 and amide group hydrogen atoms in 0th and 1st generation poly (amidoamine) dendrimers in chloroform. The average errors in the absolute and relative association constants, as predicted with the calculations, are 14.1% and 10.8%, respectively, which translate into ca. 0.08 kcal/mol and 0.06 kcal/mol errors in the absolute and relative binding free energies. We believe that this level of accuracy proves the applicability of the OPLS-AA, force field, in combination with the direct RDF integration, to reproducing and predicting absolute intermolecular association constants of low magnitudes (ca. 0.2 -- 2.0 range).

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium and 2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin. PMID:26837620

  1. 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.

  2. Functional binding surface of a β-hairpin VEGF receptor targeting peptide determined by NMR spectroscopy in living cells.

    PubMed

    Diana, Donatella; Russomanno, Anna; De Rosa, Lucia; Di Stasi, Rossella; Capasso, Domenica; Di Gaetano, Sonia; Romanelli, Alessandra; Russo, Luigi; D'Andrea, Luca D; Fattorusso, Roberto

    2015-01-02

    In this study, the functional interaction of HPLW peptide with VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) was determined by using fast (15)N-edited NMR spectroscopic experiments. To this aim, (15)N uniformly labelled HPLW has been added to Porcine Aortic Endothelial Cells. The acquisition of isotope-edited NMR spectroscopic experiments, including (15)N relaxation measurements, allowed a precise characterization of the in-cell HPLW epitope recognized by VEGFR2.

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

    PubMed

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

    2017-09-11

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

  4. NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein beta-TrCP.

    PubMed

    Evrard-Todeschi, Nathalie; Gharbi-Benarous, Josyane; Bertho, Gildas; Coadou, Gaël; Megy, Simon; Benarous, Richard; Girault, Jean-Pierre

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.

  5. /sup 1/H NMR aromatic spectrum of the operator binding domain of the l repressor: resonance assignment with application to structure and dynamics

    SciTech Connect

    Weiss, M.A.; Karplus, M.; Sauer, R.T.

    1987-02-10

    The aromatic /sup 1/H NMR resonances of the operator binding domain of lambda repressor are completely assigned. Since the resonances of this 23-kilodalton domain are too broad for the application of two-dimensional strategies for sequence-specific assignment, an alternative approach has been used. Assignments are obtained by a combination of one- and two-dimensional NMR methods, by the study of genetically altered domains, and by the biosynthetic incorporation of deuterium labels. The resulting assignments provide sensitive markers for tertiary and quaternary structure. Nuclear Overhauser enhancements demonstrate that the major features of the crystal structure, including the dimer contacts, are retained in solution. The rates of aromatic ring rotation indicate the globular domain is not rigid; significant barriers to ring rotation are observed only in the dimer contact.

  6. Mechanism of binding of the radiosensitizers metronidazole and misonidazole (RO-07-0582) to bovine and human serum albumin: a proton NMR study

    SciTech Connect

    Sulkowska, A.; Lubas, B.; Wilczok, T.

    1981-01-01

    High-resolution proton NMR spectra of the radiosensitizer metronidazole and its derivative misonidazole (RO-07-0582) were measured in D/sub 2/O at resonance frequency 60 MHz and interpreted in the aliphatic and aromatic regions. The linewidths of the NMR peaks attributed to individual fragments of nitroimidazole molecules were then analyzed in the presence of bovine and human serum albumin. With increasing concentration of serum albumin, a selectively larger broadening of the lines attributable to the protons of the aliphatic moieties than of those of the imidazole rings was observed for both compounds. This broadening for misonidazole strongly depends on the ionic strength of the solution. The results indicate a specific immobilization of the molecules of both radiosensitizers during their interaction with serum albumin and the involvement of the aliphatic chains of misonidazole and metronidazole as the primary binding sites.

  7. 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. (c) 2008 Wiley Periodicals, Inc.

  8. 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

  9. Aqueous dissociation constants of bile pigments and sparingly soluble carboxylic acids by 13C NMR in aqueous dimethyl sulfoxide: effects of hydrogen bonding.

    PubMed

    Trull, F R; Boiadjiev, S; Lightner, D A; McDonagh, A F

    1997-06-01

    pKas for the acid dissociation of the carboxyl groups of bilirubin in water have been reported recently to be 8.1-8.4, or higher. These high values were attributed to intramolecular hydrogen bonding. They have led to suggestions that monoanions of bilirubin predominate at physiologic pH and are the species transported most readily into hepatocytes by carriers. Such high aqueous pKas are inconsistent with recent 13C nuclear magnetic resonance (NMR) measurements on mesobilirubin XIII alpha, done on aqueous solutions containing dimethyl sulfoxide. To investigate whether the presence of dimethyl sulfoxide leads to unreliable values when using 13C NMR spectroscopy to determine pKas of carboxylic acids that can undergo intramolecular hydrogen bonding, we measured the pKas of 13C-labeled fumaric, maleic, and phthalic acids in solutions containing up to 27 vol% dimethyl sulfoxide. In addition, we used 13C NMR to estimate the pKas of 2,2'-methylenebis[5-carbomethoxy-4-methylpyrrole-3-[1-13C] propanoic acid], a model for the two central rings of bilirubin. Our results show that 13C NMR of aqueous dimethyl sulfoxide solutions can be used with confidence to measure pKas of intramolecularly hydrogen-bonded carboxylic acids. They support our previous estimates for the pKas of bilirubin and confirm that intramolecular hydrogen bonding has little effect on the acidity of bilirubins in water. Together with previous studies and chemical arguments they strongly suggest that reported aqueous pKas of > 8, or even > 6, for the carboxyl groups of bilirubin are incorrect and that arguments used to rationalize them are questionable.

  10. Impact of chlorpromazine self-association on its apparent binding constants with cyclodextrins: Effect of SBE(7)-beta-CD on the disposition of chlorpromazine in the rat.

    PubMed

    McIntosh, Michelle P; Leong, Nathania; Katneni, Kasiram; Morizzi, Julia; Shackleford, David M; Prankerd, Richard J

    2010-07-01

    Chlorpromazine is an antipsychotic agent with poor aqueous solubility. Complexation with SBE(7)-beta-CD can aid intravenous delivery through increasing the apparent solubility of chlorpromazine. However, chlorpromazine has also been known to self-associate. This self-association can influence its capacity to interact with other chemical species, such as cyclodextrins. This study aimed to characterise the self-association and cyclodextrin binding properties of chlorpromazine, and the effect on pharmacokinetic parameters in rats when dosed with a SBE(7)-beta-CD containing formulation. Pharmacokinetic studies of chlorpromazine in the presence and absence of SBE(7)-beta-CD were undertaken in rats. The binding constant of SBE(7)-beta-CD and chlorpromazine was studied relative to chlorpromazine concentration via fluorescence. The self-association of chlorpromazine was studied by fluorescence and UV-visible spectrophotometry. Urinary excretion of intact chlorpromazine increased in the presence of SBE(7)-beta-CD. The SBE(7)-beta-CD binding constant of chlorpromazine is highly concentration dependent and the variation can be attributed to the self-association of chlorpromazine. The apparent binding constant of chlorpromazine is highest at pharmacologically relevant concentrations, providing an explanation for the significant increase in renal chlorpromazine excretion observed in rats. (c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association

  11. Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins.

    PubMed

    Matsuyama, Bruno Y; Krasteva, Petya V; Navarro, Marcos V A S

    2017-01-01

    Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results.

  12. Specific Binding of Adamantane Drugs and Direction of their Polar Amines in the Pore of the Influenza M2 Transmembrane Domain in Lipid Bilayers and Dodecylphosphocholine Micelles Determined by NMR Spectroscopy

    PubMed Central

    Cady, Sarah D.; Wang, Jun; Wu, Yibing; DeGrado, William F.; Hong, Mei

    2011-01-01

    The transmembrane domain of the influenza M2 protein (M2TM) forms a tetrameric proton channel important for the virus lifecycle. The proton-channel activity is inhibited by amine-containing adamantyl drugs amantadine and rimantadine, which have been shown to bind specifically to the pore of M2TM near Ser31. However, whether the polar amine points to the N- or C-terminus of the channel has not yet been determined. Elucidating the polar group direction will shed light on the mechanism by which drug binding inhibits this proton channel and will facilitate rational design of new inhibitors. In this study, we determine the polar amine direction using M2TM reconstituted in lipid bilayers as well as DPC micelles. 13C-2H rotational-echo double-resonance NMR experiments of 13C-labeled M2TM and methyl-deuterated rimantadine in lipid bilayers showed that the polar amine pointed to the C-terminus of the channel, with the methyl group close to Gly34. Solution NMR experiments of M2TM in dodecylphosphocholine (DPC) micelles indicate that drug binding causes significant chemical shift perturbations of the protein that are very similar to those seen for M2TM and M2(18–60) bound to lipid bilayers. Specific 2H-labeling of the drugs permitted the assignment of drug-protein cross peaks, which indicate that amantadine and rimantadine bind to the pore in the same fashion as for bilayer-bound M2TM. These results strongly suggest that adamantyl inhibition of M2TM is achieved not only by direct physical occlusion of the pore but also by perturbing the equilibrium constant of the proton-sensing residue His37. The reproduction of the pharmacologically relevant specific pore-binding site in DPC micelles, which was not observed with a different detergent, DHPC, underscores the significant influence of the detergent environment on the functional structure of membrane proteins. PMID:21381693

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  14. 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

  15. 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

  16. Natural abundance 15N NMR by dynamic nuclear polarization: fast analysis of binding sites of a novel amine-carboxyl-linked immobilized dirhodium catalyst.

    PubMed

    Gutmann, Torsten; Liu, Jiquan; Rothermel, Niels; Xu, Yeping; Jaumann, Eva; Werner, Mayke; Breitzke, Hergen; Sigurdsson, Snorri T; Buntkowsky, Gerd

    2015-02-23

    A novel heterogeneous dirhodium catalyst has been synthesized. This stable catalyst is constructed from dirhodium acetate dimer (Rh2(OAc)4) units, which are covalently linked to amine- and carboxyl-bifunctionalized mesoporous silica (SBA-15-NH2-COOH). It shows good efficiency in catalyzing the cyclopropanation reaction of styrene and ethyl diazoacetate (EDA) forming cis- and trans-1-ethoxycarbonyl-2-phenylcyclopropane. To characterize the structure of this catalyst and to confirm the successful immobilization, heteronuclear solid-state NMR experiments have been performed. The high application potential of dynamic nuclear polarization (DNP) NMR for the analysis of binding sites in this novel catalyst is demonstrated. Signal-enhanced (13)C CP MAS and (15)N CP MAS techniques have been employed to detect different carboxyl and amine binding sites in natural abundance on a fast time scale. The interpretation of the experimental chemical shift values for different binding sites has been corroborated by quantum chemical calculations on dirhodium model complexes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Insights into the Binding of Cyclic RGD Peptidomimetics to α5β1 Integrin by using Live‐Cell NMR And Computational Studies

    PubMed Central

    Guzzetti, Ileana; Civera, Monica; Vasile, Francesca; Arosio, Daniela; Tringali, Cristina; Piarulli, Umberto; Gennari, Cesare; Pignataro, Luca

    2016-01-01

    Abstract The interaction of a small library of cyclic DKP–RGD peptidomimetics with α5β1 integrin has been investigated by means of an integrated experimental and computational approach. Bioaffinity NMR techniques, including saturation transfer difference (STD) and transferred NOESY, were applied to the ligands in a suspension of intact MDA‐MB‐231 breast cancer cells, in which integrin α5β1 is highly expressed. The NMR data were compared with the docking calculations of the RGD ligands in the crystal structure of the α5β1 binding site, and were integrated with competitive binding assays to the purified α5β1 integrin. Ligand binding epitopes involve protons of both the RGD moiety and the DKP scaffold, although the stereochemistry and the functionalization of the DKP scaffold as well as the macrocycle conformation determine a great variability in the interaction. The ligand showing the highest number of STD signals is also the most potent α5β1 ligand of the series, displaying a nanomolar IC 50 value. PMID:28168158

  18. 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.

  19. 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.

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

    PubMed

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

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1996-05-01

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

  2. High-Resolution Solid-State NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica Matrix.

    PubMed

    Cerofolini, Linda; Giuntini, Stefano; Louka, Alexandra; Ravera, Enrico; Fragai, Marco; Luchinat, Claudio

    2017-08-31

    Solid-state NMR is becoming a powerful tool to detect atomic-level structural features of biomolecules even when they are bound to (or trapped in) solid systems that lack long-range three-dimensional order. We here demonstrate that it is possible to probe protein-ligand interactions from a protein-based perspective also when the protein is entrapped in silica, thus translating into biomolecular solid-state NMR all of the considerations that are usually made to understand the chemical nature of the interaction of a protein with its ligands. This work provides a proof of concept that also immobilized enzymes can be used for protein-based NMR protein-ligand interactions for drug discovery.

  3. DOSY NMR and MALDI-TOF evidence of covalent binding the DNA duplex by trimethylammonium salts of topotecan upon near UV irradiation.

    PubMed

    Naumczuk, Beata; Hyz, Karolina; Kawęcki, Robert; Bocian, Wojciech; Bednarek, Elżbieta; Sitkowski, Jerzy; Wielgus, Ewelina; Kozerski, Lech

    2015-08-01

    Using DOSY NMR and MALDI-TOF MS techniques, we present evidence that quaternary trimethylammonium salts of topotecan, [TPT-NMe3 ](+) X(-) (X = CF3SO3, HCOO), bind covalently the natural DNA oligomer upon near UV irradiation in water under physiological conditions. It is shown that formate salt is very reactive at pH 7 and requires short irradiation time. This weak irradiation at 365 nm paves the way for a new application of TPT derivatives in clinical use, which can dramatically increase the therapeutic effects of a medicine.

  4. Binding.

    ERIC Educational Resources Information Center

    Rebsamen, Werner

    1981-01-01

    Categorizes contemporary methods of binding printed materials in terms of physical preservation--hand binding (archival restoration), edition binding (paperback, hardcover), publication binding (magazines), textbook binding (sidesewn), single-sheet binding (loose-leaf, mechanical), and library binding (oversewn, sidesewn). Seven references are…

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  6. 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.

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

    PubMed

    Live, D H; Cowburn, D; Breslow, E

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H 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 of 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 neurophysin 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 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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.

  9. A novel 2-oxoindolinylidene inhibitor of bacterial MurD ligase: Enzyme kinetics, protein-inhibitor binding by NMR and a molecular dynamics study.

    PubMed

    Simčič, Mihael; Pureber, Kaja; Kristan, Katja; Urleb, Uroš; Kocjan, Darko; Grdadolnik, Simona Golič

    2014-08-18

    N-(5-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)4-oxo-2-thioxo-1,3-thiazolidin-3-yl)nicotinamide, a 2-oxoindolinylidene derivative with novel structure scaffold, was evaluated for inhibition potency against the MurD enzyme from Escherichia coli using an enzyme steady-state kinetics study. The compound exerted competitive inhibition with respect to UMA, a MurD substrate, and affected bacterial growth. Furthermore, we isolated and purified (13)C selectively labeled MurD enzyme from E. coli and evaluated the binding interactions of the new compound using the (1)H/(13)C-HSQC 2D NMR method. Molecular dynamics calculations showed stable structure for the MurD-inhibitor complex. The binding mode of novel inhibitor was determined and compared to naphthalene-N-sulfonamide-d-Glu derivatives, transition state mimicking inhibitors, UMA and AMP-PCP, an ATP analog. It binds to the UDP/MurNAc binding region. In contrast to transition state mimicking inhibitors, it does not interact with the enzyme's C-terminal domain, which can be beneficial for ligand binding. A pharmacophore pattern was established for the design of novel drugs having a propensity to inhibit a broad spectrum of Mur enzymes.

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

    NASA Astrophysics Data System (ADS)

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

    2017-10-01

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

  11. 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).

  12. NMR study suggests a major role for Arg111 in maintaining the structure and dynamical properties of type II human cellular retinoic acid binding protein.

    PubMed

    Wang, L; Yan, H

    1998-09-15

    The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 phi dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.26 and 0.98 +/- 0.23 A for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19-Ala36, Glu73-Cys81, and Leu99-Pro105) clustered around the ligand entrance more than 17 A away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII.

  13. A simple method for estimating equilibrium constants for serum testosterone binding resulting in an optimal free testosterone index for use in elderly men.

    PubMed

    Ross, H Alec; Meuleman, Eric J; Sweep, Fred C G J

    2005-01-01

    An algorithm was developed to evaluate equilibrium constants for testosterone (Te) and sex hormone-binding globulin (SHBG) or albumin from serum free testosterone (FTe) measurements performed in a panel of 30 healthy elderly men by means of a near-reference method, i.e., symmetric dialysis (affinity constants: SHBG-Te, 1.13 x 10(9) L/mol; albumin-Te, 4.4 x 10(4) L/mol). Using these estimates, a free testosterone index (FTeI) was calculated from total Te and SHBG concentrations in a further 35 elderly men. This FTeI perfectly matches with actually measured free testosterone concentrations by symmetric dialysis in this second group, with a mean ratio index/measurement of 0.998+/-0.016 (SEM). The efficacy of the algorithm, which represents a simple alternative to previous cumbersome methods for estimation of equilibrium constants, is thereby demonstrated.

  14. [13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.

    PubMed Central

    He, Q Y; Mason, A B; Tam, B M; MacGillivray, R T; Woodworth, R C

    1999-01-01

    The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized. PMID:10585877

  15. 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).

  16. 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

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

    PubMed Central

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

    2010-01-01

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

  18. In Depth Analysis on the Binding Sites of Adamantane Derivatives in HCV (Hepatitis C Virus) p7 Channel Based on the NMR Structure

    PubMed Central

    Du, Qi-Shi; Wang, Shu-Qing; Chen, Dong; Meng, Jian-Zong; Huang, Ri-Bo

    2014-01-01

    Background The recently solved solution structure of HCV (hepatitis C virus) p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine) was determined in a hydrophobic pocket. However the pharmocophore (−NH2) of the ligand was not assigned a specific binding site. Results The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T)/6–311+G(d,p) and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed. Conclusions Some useful viewpoints are concluded as follows. (1) The amino group (−NH2) of adamantane derivatives is protonated (−NH3+), and the positively charged cation may form cation-π interactions with aromatic amino acids. (2) The aromatic amino acids (His17, Phe20, and Trp21) are the possible binding sites for the protonated amino group (−NH3+) of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3) The higher inhibition potent of rimantadine than amantadine probably because of its higher pKa value (pKa = 10.40) and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed. PMID:24714586

  19. BDflex: A method for efficient treatment of molecular flexibility in calculating protein-ligand binding rate constants from Brownian dynamics simulations

    NASA Astrophysics Data System (ADS)

    Greives, Nicholas; Zhou, Huan-Xiang

    2012-10-01

    A method developed by Northrup et al. [J. Chem. Phys. 80, 1517 (1984)], 10.1063/1.446900 for calculating protein-ligand binding rate constants (ka) from Brownian dynamics (BD) simulations has been widely used for rigid molecules. Application to flexible molecules is limited by the formidable computational cost to treat conformational fluctuations during the long BD simulations necessary for ka calculation. Here, we propose a new method called BDflex for ka calculation that circumvents this problem. The basic idea is to separate the whole space into an outer region and an inner region, and formulate ka as the product of kE and bar η _d, which are obtained by separately solving exterior and interior problems. kE is the diffusion-controlled rate constant for the ligand in the outer region to reach the dividing surface between the outer and inner regions; in this exterior problem conformational fluctuations can be neglected. bar η _d is the probability that the ligand, starting from the dividing surface, will react at the binding site rather than escape to infinity. The crucial step in reducing the determination of bar η _d to a problem confined to the inner region is a radiation boundary condition imposed on the dividing surface; the reactivity on this boundary is proportional to kE. By confining the ligand to the inner region and imposing the radiation boundary condition, we avoid multiple-crossing of the dividing surface before reaction at the binding site and hence dramatically cut down the total simulation time, making the treatment of conformational fluctuations affordable. BDflex is expected to have wide applications in problems where conformational fluctuations of the molecules are crucial for productive ligand binding, such as in cases where transient widening of a bottleneck allows the ligand to access the binding pocket, or the binding site is properly formed only after ligand entrance induces the closure of a lid.

  20. SedNMR: on the edge between solution and solid-state NMR.

    PubMed

    Bertini, Ivano; Luchinat, Claudio; Parigi, Giacomo; Ravera, Enrico

    2013-09-17

    Solid-state NMR (SS-NMR) of proteins requires that those molecules be immobilized, usually by crystallization, freezing, or lyophilization. However, self-crowding can also slow molecular rotation sufficiently to prevent the nuclear interactions from averaging. To achieve self-crowding, researchers can use a centrifugal field to create a concentration gradient or use regular ultracentrifugation to produce highly concentrated, gel-like solutions. Thus sedimented solute NMR (SedNMR) provides a simple method to prepare biological samples for SS-NMR experiments with minimal perturbation. This method may also give researchers a way to investigate species that are not otherwise accessible by NMR. We induce the sedimentation in one of two ways: (1) by the extreme centrifugal force exerted during magic angle spinning (MAS-induced sedimentation or in situ) or (2) by an ultracentrifuge (UC-induced sedimentation or ex situ). Sedimentation is particularly useful in situations where it is difficult to obtain protein crystals. Furthermore, because the proteins remain in a largely hydrated state, the sedimented samples may provide SS-NMR spectra that have better resolution than the spectra from frozen solutions or lyophilized powders. If sedimentation is induced in situ, the same protein sample can be used for both solution and SS-NMR studies. Finally, we show that in situ SedNMR can be used to detect the NMR signals of large molecular adducts that have binding constants that are too weak to allow for the selective isolation and crystallization of the complexed species. We can selectively induce sedimentation for the heaviest molecular species. Because the complexed molecules are subtracted from the bulk solution, the reaction proceeds further toward the formation of complexes.

  1. The effects of the CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography

    PubMed Central

    Charpentier, Thomas H.; Thompson, Laura E.; Liriano, Melissa A.; Varney, Kristen M.; Wilder, Paul T.; Pozharski, Edwin; Toth, Eric A.; Weber, David J.

    2010-01-01

    Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca2+-S100B and a target, TRTK-12, which binds to the p53 binding site on S100B. The structures of Ca2+-S100B (1.5 Å resolution) and S100B-Ca2+-TRTK12 (2.0 Å resolution) determined here indicate that the S100B-Ca2+-TRTK12 complex is dominated by an interaction between Trp-7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca2+-S100B involving residues in helices 2 & 3 and loop 2. As with a S100B-Ca2+-p53 peptide complex, TRTK-12 binding to Ca2+-S100B was found to increase the proteins Ca2+ ion binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca2+ ligands and/or improved Ca2+ ion coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca2+-TRTK12 and S100B-Ca2+ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca2+-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR 15N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca2+-S100B. Such a loss of protein motion may also provide an explanation for how calcium ion binding affinity is increased upon binding a target. Lastly, it follows that any small molecule inhibitor bound to Ca2+-S100B would also have to cause an increase in calcium ion binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug-design studies involving S100B. PMID:20053360

  2. Solution NMR structure of RHE_CH02687 from Rhizobium etli: A novel flavonoid-binding protein.

    PubMed

    Liang, Chunjie; Zhu, Jiang; Hu, Rui; Ramelot, Theresa A; Kennedy, Michael A; Liu, Maili; Yang, Yunhuang

    2017-05-01

    We report the solution NMR structure of RHE_CH02687 from Rhizobium etli. Its structure consists of two β-sheets that together with two short and one long α-helix form a hydrophobic cavity. This protein shows a high structural similarity to the prokaryotic protein YndB from Bacillus subtilis, and the eukaryotic protein Aha1. NMR titration experiments confirmed that RHE_CH02687, like its homolog YndB, interacted with flavonoids, giving support for a biological function as a flavonoid sensor in the symbiotic interaction between R. etli and plants. In addition, our study showed no evidence for a direct interaction between RHE_CH02687 and HtpG, the R. etli homolog of Hsp90. Proteins 2017; 85:951-956. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Substrate Induced Conformational Studies of the Hormone Binding Domain of the Human Estrogen Receptor by Fluorine NMR

    DTIC Science & Technology

    1998-07-01

    our previous investigation based on Trp absorbance and fluorescence spectroscopy ( Hasselbacher et al., 1995a), showing that the NMR and optical... Hasselbacher et al., 1995a). Following standard protocols (Sambrook et al., 1989), the plasmids were transformed into the E. coli tryptophan auxotroph... Hasselbacher et al., 1995a). With the shorter induction period used here, most of the expressed protein is retained in the cells. Therefore, the proteins

  4. 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;…

  5. 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;…

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

    PubMed

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

    2017-05-23

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

  7. Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking.

    PubMed

    Weidel, Elisabeth; de Jong, Johannes C; Brengel, Christian; Storz, Michael P; Braunshausen, Andrea; Negri, Matthias; Plaza, Alberto; Steinbach, Anke; Müller, Rolf; Hartmann, Rolf W

    2013-08-08

    Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.

  8. NMR structure of a heterodimeric SAM:SAM complex: characterization and manipulation of EphA2 binding reveal new cellular functions of SHIP2.

    PubMed

    Lee, Hyeong J; Hota, Prasanta K; Chugha, Preeti; Guo, Hong; Miao, Hui; Zhang, Liqun; Kim, Soon-Jeung; Stetzik, Lukas; Wang, Bing-Cheng; Buck, Matthias

    2012-01-11

    The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural basis for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex toward alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM, whereas EphB members may not; correspondingly, we demonstrate binding of EphA1, but not of EphB2, to SHIP2. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting receptor coordinated chemotactic cell migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. 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.

  10. 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.

  11. Semi-empirical spectrophotometric (SESp) method for the indirect determination of the ratio of cationic micellar binding constants of counterions X⁻ and Br⁻(K(X)/K(Br)).

    PubMed

    Khan, Mohammad Niyaz; Yusof, Nor Saadah Mohd; Razak, Norazizah Abdul

    2013-01-01

    The semi-empirical spectrophotometric (SESp) method, for the indirect determination of ion exchange constants (K(X)(Br)) of ion exchange processes occurring between counterions (X⁻ and Br⁻) at the cationic micellar surface, is described in this article. The method uses an anionic spectrophotometric probe molecule, N-(2-methoxyphenyl)phthalamate ion (1⁻), which measures the effects of varying concentrations of inert inorganic or organic salt (Na(v)X, v = 1, 2) on absorbance, (A(ob)) at 310 nm, of samples containing constant concentrations of 1⁻, NaOH and cationic micelles. The observed data fit satisfactorily to an empirical equation which gives the values of two empirical constants. These empirical constants lead to the determination of K(X)(Br) (= K(X)/K(Br) with K(X) and K(Br) representing cationic micellar binding constants of counterions X and Br⁻). This method gives values of K(X)(Br) for both moderately hydrophobic and hydrophilic X⁻. The values of K(X)(Br), obtained by using this method, are comparable with the corresponding values of K(X)(Br), obtained by the use of semi-empirical kinetic (SEK) method, for different moderately hydrophobic X. The values of K(X)(Br) for X = Cl⁻ and 2,6-Cl₂C6H₃CO₂⁻, obtained by the use of SESp and SEK methods, are similar to those obtained by the use of other different conventional methods.

  12. Binding modes and dynamics of distamycin A with several A-T-rich oligomers by two-dimensional NMR

    SciTech Connect

    Pelton, J.G.

    1990-01-01

    Nuclear Overhauser effect spectroscopy has been used to identify the binding modes and to probe the dynamics of distamycin A in complexes with d(CGCGAATTCGCG){sub 2}, d(CGCAAATTGGC):d(GCCAATTTGCG), and d(CGCAAATTTGCG){sub 2}. Distamycin A binds to the minor-groove of the AATT segment of the d(CGCGAATTCGCG){sub 2} duplex. The binding site is characterized by drug-DNA van der Waals contacts, three-center hydrogen bonds, and stacking of sugar O4{prime} atoms over each of the drug pyrrole rings. The drugs are in close contact with the DNA and one another. Distamycin A binds in one-drug and symmetric two-drug binding modes with d(CGCAAATTTGCG){sub 2}. Energy refinement suggests that electrostatic interactions, intermolecular hydrogen bonds, and stacking interactions contribute to stabilization of these complexes. 80 refs., 38 figs., 14 tabs.

  13. NMR Structure of Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1): Implications for pH-Sensitive Pheromone Detection†

    PubMed Central

    Xu, Xianzhong; Xu, Wei; Rayo, Josep; Ishida, Yuko; Leal, Walter S.; Ames, James B.

    2010-01-01

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from Amyelois transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multi-angle light scattering, and 15N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven α-helices (α1: L8-L23, α2: D27-F36, α3: R46-V62, α4: A73-M78; α5: D84-S100; α6: R107-L125; α7: M131-E141) that adopt an overall main chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108 and C97/C117, and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141 and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (α7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139 and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions. PMID:20088570

  14. 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.

    PubMed

    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, (n)J(CF)- and (n)J(FF)-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 (n)J(CF)- and (n)J(FF)-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

  15. 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.

  16. Estimation of binding rate constants using a simultaneous mixed-effects method: application to monoamine transporter reuptake inhibitor reboxetine

    PubMed Central

    Benson, Neil; Snelder, Nelleke; Ploeger, Bart; Napier, Carolyn; Sale, Harriet; JM Birdsall, Nigel; Butt, Richard P; van der Graaf, Piet H

    2010-01-01

    Background and purpose: Reboxetine is a clinically used antidepressant and is a racemic mixture of two enantiomers, SS- and RR-reboxetine. The aim of the work described in this manuscript was to determine the kinetics of binding of the RR- and SS-reboxetine to the human noradrenaline transporter (hNET). Experimental approach: We have applied a simultaneous mixed-effects method to the analysis of the transient kinetics of binding of SS-, RR- and racemic reboxetine to hNET. This method allowed simultaneous modelling of multiple datasets, taking into account inter-experiment variability, thereby facilitating robust parameter estimation and minimizing the assumptions made. Key results: The mixed-effects method proved simple and robust. SS-reboxetine bound to hNET according to a one-step binding model with the SS-enantiomer having 130-fold higher steady state affinity than the RR-enantiomer (Kd= 0.076 ± 0.009 nM vs. 9.7 ± 0.8 nM respectively). The kon for SS-reboxetine was c. 1.4 × 105 M−1·s−1 and koff 1.05 × 10−5 s−1 (t1/2∼18 h). The kon for RR-reboxetine was c. 4.3 × 105 M−1·s−1 and koff 4.2 × 10−3 s−1 (t1/2∼3 min). The racemate behaved as expected for an equimolar mixture of RR- and SS-reboxetine, assuming mutually exclusive binding. Conclusions and implications: These data will be useful for the interpretation of the behaviour of reboxetine and its enantiomers in man and the method used could be applied to other candidate drugs. PMID:20423348

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

    PubMed

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

    2016-12-01

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

  18. 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.

  19. 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

  20. 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

  1. Combining NMR spectral and structural data to form models of polychlorinated dibenzodioxins, dibenzofurans, and biphenyls binding to the AhR

    NASA Astrophysics Data System (ADS)

    Beger, Richard D.; Buzatu, Dan A.; Wilkes, Jon G.

    2002-10-01

    A three-dimensional quantitative spectrometric data-activity relationship (3D-QSDAR) modeling technique which uses NMR spectral and structural information that is combined in a 3D-connectivity matrix has been developed. A 3D-connectivity matrix was built by displaying all possible assigned carbon NMR chemical shifts, carbon-to-carbon connections, and distances between the carbons. Two-dimensional 13C-13C COSY and 2D slices from the distance dimension of the 3D-connectivity matrix were used to produce a relationship among the 2D spectral patterns for polychlorinated dibenzofurans, dibenzodioxins, and biphenyls (PCDFs, PCDDs, and PCBs respectively) binding to the aryl hydrocarbon receptor (AhR). We refer to this technique as comparative structural connectivity spectral analysis (CoSCoSA) modeling. All CoSCoSA models were developed using forward multiple linear regression analysis of the predicted 13C NMR structure-connectivity spectral bins. A CoSCoSA model for 26 PCDFs had an explained variance (r2) of 0.93 and an average leave-four-out cross-validated variance (q4 2) of 0.89. A CoSCoSA model for 14 PCDDs produced an r2 of 0.90 and an average leave-two-out cross-validated variance (q2 2) of 0.79. One CoSCoSA model for 12 PCBs gave an r2 of 0.91 and an average q2 2 of 0.80. Another CoSCoSA model for all 52 compounds had an r2 of 0.85 and an average q4 2 of 0.52. Major benefits of CoSCoSA modeling include ease of development since the technique does not use molecular docking routines.

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

    PubMed Central

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

    2016-01-01

    Abstract 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

  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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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.

  5. NMR Identification of the Binding Surfaces Involved in the Salmonella and Shigella Type III Secretion Tip-Translocon Protein-Protein Interactions

    PubMed Central

    McShan, Andrew C.; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M.; De Guzman, Roberto N.

    2017-01-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. PMID:27093649

  6. 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.

  7. Mapping the Binding Modes of Hemilabile Pincer-Crown Ether Ligands in Solution Using Diamagnetic Anisotropic Effects on NMR Chemical Shift.

    PubMed

    Camp, Andrew M; Kita, Matthew R; Grajeda, Javier; White, Peter S; Dickie, Diane A; Miller, Alexander J M

    2017-09-05

    A protocol for identifying ligand binding modes in a series of iridium pincer complexes bearing hemilabile aza-crown ether ligands has been developed using readily accessible NMR methods. The approach was tested on a collection of 13 structurally diverse pincer-crown ether complexes that include several newly characterized species. New synthetic routes enable facile interconversion of coordination modes and supporting ligands. Detailed structural assignments of five complexes reveal that the difference in chemical shift (Δδ) between geminal protons in the crown ether is influenced by diamagnetic anisotropy arising from halides and other ligands in the primary coordination sphere. The average difference in chemical shift between diastereotopic geminal protons in the crown ether macrocycle (Δδavg), as determined through a single (1)H-(13)C HSQC experiment, provides information on the pincer ligand binding mode by establishing whether the macrocycle is in close proximity to the metal center. The Δδavg values for binding modes that involve chelating ether(s) bound to iridium are roughly 2-fold larger than those for tridentate complexes with no Ir-O bonds.

  8. NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

    PubMed

    McShan, Andrew C; Kaur, Kawaljit; Chatterjee, Srirupa; Knight, Kevin M; De Guzman, Roberto N

    2016-08-01

    The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Protein Structural Studies by Paramagnetic Solid-State NMR Spectroscopy Aided by a Compact Cyclen-Type Cu(II) Binding Tag

    PubMed Central

    Sengupta, Ishita; Gao, Min; Arachchige, Rajith J.; Nadaud, Philippe S.; Cunningham, Timothy F.; Saxena, Sunil; Schwieters, Charles D.; Jaroniec, Christopher P.

    2014-01-01

    Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu2+ binding tag, 1-(2-(pyridin-2-yldisulfanyl)ethyl)-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC-Cu2+ K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu2+ center experience considerable transverse PREs leading to severely attenuated resonances in 2D 15N-13C correlation spectra. For more distant residues, electron-nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for 15N-Cu2+ distances up to ~20 Å. PMID:25432438

  10. 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.

  11. Reduced Flavin: NMR investigation of N(5)-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst

    PubMed Central

    Macheroux, Peter; Ghisla, Sandro; Sanner, Christoph; Rüterjans, Heinz; Müller, Franz

    2005-01-01

    Background The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base. PMID:16309555

  12. NMR studies of the POU-specific DNA-binding domain of Oct-1: sequential 1H and 15N assignments and secondary structure.

    PubMed

    Cox, M; Dekker, N; Boelens, R; Verrijzer, C P; van der Vliet, P C; Kaptein, R

    1993-06-15

    The 1H and 15N resonances of the POU-specific DNA-binding domain of transcription factor Oct-1 have been assigned sequentially using two-dimensional homo- and heteronuclear NMR techniques, as well as three-dimensional heteronuclear NMR techniques, including TOCSY, 2D NOE, and NOESY-HMQC experiments. A number of typical short- and medium-range NOE contacts, as well as amide proton exchange data, gave evidence for the presence of four alpha-helices, in the peptide segments 1-19, 23-34, 40-49, and 54-71, which are connected by short loops of irregular structure. Interestingly, the second helix contains three glycine residues and the fourth helix a proline in the middle of the helix. Although the regular pattern of hydrogen bonds in the fourth helix is interrupted, due to the absence of an amide proton in proline, the helix is remarkably stable. All four helices are amphipathic, which suggests a packing of the apolar sides of the helices in the folded structure of the protein.

  13. 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.

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

    PubMed

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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.

  16. 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.

  17. 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.

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

    PubMed

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

    2014-10-21

    We discuss the effect of electron correlation on the unexpected differential sensitivity (UDS) in the (1)J(C-H) coupling constant of CH4 using a decomposition into contributions from localized molecular orbitals and compare with the (1)J(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.

  19. Structural Determinants in the Binding of BB2 Receptor Ligands: In Silico, X-Ray and NMR Studies in PD176252 Analogues.

    PubMed

    Carrieri, Antonio; Lacivita, Enza; Belviso, Benny Danilo; Caliandro, Rocco; Mastrorilli, Piero; Gallo, Vito; Niso, Mauro; Leopoldo, Marcello

    2017-01-01

    The mammalian bombesin receptor family comprises three G proteincoupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    PubMed

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

    2016-12-15

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

  1. 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

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

    PubMed

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

    2017-06-12

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

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

    PubMed

    Konuma, Tsuyoshi; Harada, Erisa; Sugase, Kenji

    2015-12-01

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

  4. 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

  5. Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

    PubMed Central

    Hensmann, M.; Booker, G. W.; Panayotou, G.; Boyd, J.; Linacre, J.; Waterfield, M.; Campbell, I. D.

    1994-01-01

    The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study. PMID:7522724

  6. Effect of serum albumin presence on the binding constant of metronidazole to the phospholipid membranes fluorescence study

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.

    1999-05-01

    The experiment was designed to test the possibility of entrapping the drug metronidazole into liposome vesicles in order to use liposome vesicles as a drug carrier. We estimated the effect of size of the liposome and the presence of serum albumin on the leakage of the drug. Some interactions between the lipid membrane surface and serum albumin were demonstrated. As the effects of lipid composition and physical states in membranes on protein adsorption and binding are poorly understood, it is difficult to estimate the protein interaction with the lipid membrane surface. The fluorescence quenching technique was used in this investigation. The fluorescence of serum albumin tryptophanyl residues is reduced by the presence of metronidazole. When incorporated into liposomes, metronidazole reduces the fluorescence of tryptophanyl residues of BSA to a lesser extent. The least effect of metronidazole on the fluorescence of albumin is shown when the drug is incorporated into large liposomes (450 nm). Larger liposomes are less susceptible to the presence of serum albumin than the smaller ones. Large size of the liposomes is necessary to retain their stability in plasma.

  7. Manifestation of stereoelectronic effects on the calculated carbon-hydrogen bond lengths and one bond (1)J(C-H) NMR coupling constants in cyclohexane, six-membered heterocycles, and cyclohexanone derivatives.

    PubMed

    Cuevas, Gabriel; Juaristi, Eusebio

    2002-11-06

    Cyclohexane (1), oxygen-, sulfur-, and/or nitrogen-containing six-membered heterocycles 2-5, cyclohexanone (6), and cyclohexanone derivatives 7-16 were studied theoretically [B3LYP/6-31G(d,p) and PP/IGLO-III//B3LYP/6-31G(d,p) methods] to determine the structural (in particular C-H bond distances) and spectroscopic (specifically, one bond (1)J(C-H) NMR coupling constants) consequences of stereoelectronic hyperconjugative effects. The results confirm the importance of n(X) --> sigma*(C-H)(app) (where X = O, N), sigma(C-H)(ax) --> pi*(C=O), sigma(S-C) --> sigma*(C-H)(app), sigma(C-S)-->sigma*(C-H)(app), beta-n(O) --> sigma*(C-H), and sigma(C-H) --> sigma*(C-H)(app) hyperconjugation, as advanced in previous theoretical models. Calculated r(C-H) bond lengths and (1)J(C-H) coupling constants for C-H bonds participating in more than one hyperconjugative interaction show additivity of the effects.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  9. NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1.

    PubMed

    Wang, Wei; Lim, Liangzhong; Baskaran, Yohendran; Manser, Ed; Song, Jianxing

    2013-08-16

    Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-22

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

  12. Models of steroid binding based on the minimum deviation of structurally assigned 13C NMR spectra analysis (MiDSASA).

    PubMed

    Beger, Richard D; Harris, Stephen; Xie, Qian

    2004-01-01

    This paper develops a quantitative k-nearest neighbors modeling technique. The technique is used to demonstrate that a compound's biological binding activity to a receptor can be calculated from the minimum of the square root of the sum of squared deviations (SSSD) of a structurally assigned chemical shift on a template between the unknown compound to be predicted and a set of known compounds with known activities. When building models of biological activity, nonlinear relationships are built into the input training data. If a model is developed by selecting only compounds with minimum structurally assigned chemical shift deviations from the unknown compound, some of the nonlinear relationships can be removed. The smaller the total chemical shift deviation between a compound with known activity and another compound with unknown activity, the more likely it will have similar biological, chemical, and physical properties. This means that a model can be produced without rigorous statistics or neural networks. This technique is similar to structure-activity relationship (SAR) modeling, but instead of relying on substructure fragments to produce a model, this new model is based on minimum chemical shift differences on those substructure fragments. We refer to this method as minimum deviation of structurally assigned spectra analysis (MiDSASA) modeling. Modeling by the minimum deviation concept can be applied to other chemoinformatic data analyses such as metabolite concentrations in metabolic pathways for metabolomics research. A MiDSASA template model for 30 steroids binding the corticosterone binding globulin based on the activity factors of the two nearest compounds had a correlation of 0.88. A MiDSASA template model for 50 steroids binding the aromatse enzyme based on the average activity of the four nearest compounds had a correlation of 0.71.

  13. Binding studies of adhesion/growth-regulatory galectins with glycoconjugates monitored by surface plasmon resonance and NMR spectroscopy.

    PubMed

    Muñoz, F Javier; Santos, J Ignacio; Ardá, Ana; André, Sabine; Gabius, Hans-Joachim; Sinisterra, José V; Jiménez-Barbero, Jesús; Hernáiz, María J

    2010-06-28

    Functionalized fluorescent glycans have the potential to act as tools to detect and analyze protein-carbohydrate interactions. We present here a facile strategy for immobilization of functionalized lactose as a model disaccharide. Bioactivity was tested with three members of the adhesion/growth-regulatory galectins family in different types of assay, i.e. matrix in surface plasmon resonance (SPR), free ligand in solution by STD/trNOESY and docking measurements. In all cases, the activity of the disaccharide was maintained. The attachment of this new fluorescent glycoconjugate to the surface results in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The results indicate that this new glycoconjugate exhibits binding affinity to galectin-1, 3 and CG-16. Kinetic analysis of the interaction between these galectins and immobilized glycoconjugate by SPR yielded a K(D) of 1.01 mM for galectin-1, 83.5 microM for galectin-3 and 0.28 mM for CG-16. No major contacts to the aglyconic part were detected, which might compromise the specificity of the binding process with other headgroups. Thus, testing these proteins offers the potential for medical applications to detect these endogenous effectors or further derivatives and characterize their carbohydrate specificity.

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

    PubMed

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

    2003-11-12

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

  15. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. 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.

  17. Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy

    SciTech Connect

    Van Gorkom, L.C.; Horvath, L.I.; Hemminga, M.A.; Sternberg, B.; Watts, A. )

    1990-04-24

    The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.

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

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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

  19. Substituent effects on some calculated NMR data in T-shaped configuration of benzene dimer

    NASA Astrophysics Data System (ADS)

    Masoodi, Hamid Reza; Zakarianezhad, Mohammad; Bagheri, Sotoodeh; Makiabadi, Batoul; Shool, Motahareh

    2014-10-01

    Some NMR data of T-shaped configuration of benzene dimer are theoretically examined in the presence of different substituents. The geometries are optimized at MP2/6-311++G(d,p) level of theory. The NMR calculations are performed at B3LYP/6-311++G(d,p) and PBE0/6-311++G(d,p) levels. The correlation between NMR data and electron-donating (or -withdrawing) of substituents is investigated. Linear relationships between Hammett constants and some NMR data can be found with good correlation coefficients. In addition to geometrical features and binding energies, the study of the topological properties of electron charge density aids in better understanding of NMR data in these complexes.

  20. Premelting base pair opening probability and drug binding constant of a daunomycin-poly d(GCAT).poly d(ATGC) complex.

    PubMed Central

    Chen, Y Z; Prohofsky, E W

    1994-01-01

    We calculate room temperature thermal fluctuational base pair opening probability of a daunomycin-poly d(GCAT).poly d(ATGC) complex. This system is constructed at an atomic level of detail based on x-ray analysis of a crystal structure. The base pair opening probabilities are calculated from a modified self-consistent phonon approach of anharmonic lattice dynamics theory. We find that daunomycin binding substantially enhances the thermal stability of one of the base pairs adjacent the drug because of strong hydrogen bonding between the drug and the base. The possible effect of this enhanced stability on the drug inhibition of DNA transcription and replication is discussed. We also calculate the probability of drug dissociation from the helix based on the selfconsistent calculation of the probability of the disruption of drug-base H-bonds and the unstacking probability of the drug. The calculations can be used to determine the equilibrium drug binding constant which is found to be in good agreement with observations on similar daunomycin-DNA systems. PMID:8011914

  1. BDflex: A method for efficient treatment of molecular flexibility in calculating protein-ligand binding rate constants from Brownian dynamics simulations

    PubMed Central

    Greives, Nicholas; Zhou, Huan-Xiang

    2012-01-01

    A method developed by Northrup [J. Chem. Phys. 80, 1517 (1984)]10.1063/1.446900 for calculating protein-ligand binding rate constants (ka) from Brownian dynamics (BD) simulations has been widely used for rigid molecules. Application to flexible molecules is limited by the formidable computational cost to treat conformational fluctuations during the long BD simulations necessary for ka calculation. Here, we propose a new method called BDflex for ka calculation that circumvents this problem. The basic idea is to separate the whole space into an outer region and an inner region, and formulate ka as the product of kE and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}\\bar \\eta _{\\rm d}\\end{equation*} \\end{document}η¯d, which are obtained by separately solving exterior and interior problems. kE is the diffusion-controlled rate constant for the ligand in the outer region to reach the dividing surface between the outer and inner regions; in this exterior problem conformational fluctuations can be neglected. \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}\\bar \\eta _{\\rm d}\\end{equation*} \\end{document}η¯d is the probability that the ligand, starting from the dividing surface, will react at the binding site rather than escape to infinity. The crucial step in reducing the determination of \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin

  2. Competition STD NMR for the detection of high-affinity ligands and NMR-based screening.

    PubMed

    Wang, Yu-Sen; Liu, Dingjiang; Wyss, Daniel F

    2004-06-01

    The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads. Copyright 2004 John Wiley & Sons, Ltd.

  3. Equivalence of Mg2+ and Na+ ions in salt dependence of the equilibrium binding and dissociation rate constants of Escherichia coli RNA polymerase open complex.

    PubMed

    Loziński, Tomasz; Bolewska, Krystyna; Wierzchowski, Kazimierz L

    2009-06-01

    Conflicting experimental data on the influence of Mg(2+) ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of Escherichia coli RNA polymerase led us to carry systematic measurements of the dissociation rate constant (k(d)) and thermodynamic stability of complexes at lambdaP(R) and Pa promoters in a broad range of [NaCl] and [MgCl(2)] at 25, 31 and 37 degrees C, using fluorescence detected abortive transcription assay. Values of k(d) determined in MgCl(2) in the presence of heparin, as a commonly used anionic competitor, were shown to depend on heparin concentration whereas in NaCl this effect was not observed. Kinetics of dissociation was therefore determined in the course of salt-induced down-shift of the binding equilibrium. Salt derivatives of k(d)'s (n(d)) appeared to be similar in NaCl (approximately 8.5) and MgCl(2) (approximately 10) for both complexes. Isotherms of fractional occupancy of promoters by RNAP as a function of ln [salt] were shown to conform to a sigmoid Boltzman function parameterized to include binding constant of RPo and a net change (n(obs)) in the number of electrolyte ions associated with complex components upon its formation/dissociation. The fitted values of n(obs) appeared also similar in NaCl and in MgCl(2): approximately 18 for RPo/lambdaP(R) and approximately 20 for RPo/Pa, respectively. Overall unfavorable vant'Hoff enthalpy (DeltaH(obs)) of RPo proved to be much higher in MgCl(2) than in NaCl by ca. 20 kcal/mol for both complexes, rendering them profoundly less stable in the former salt. In both salts, DeltaH(obs) was higher by approximately 30 kcal/mol for RPo/Pa relative to RPo/lambdaP(R). Similarity of n(obs) and n(d) values for the two salts indicates thermodynamic equivalence of Mg(2+) and Na(+) in [salt]-controlled binding equilibrium of RPo. This finding remains in disagreement with earlier data and suggests that salt effects on open complex stability should be sought in global

  4. Probing Hydronium Ion Histidine NH Exchange Rate Constants in the M2 Channel via Indirect Observation of Dipolar-Dephased (15)N Signals in Magic-Angle-Spinning NMR.

    PubMed

    Fu, Riqiang; Miao, Yimin; Qin, Huajun; Cross, Timothy A

    2016-12-14

    Water-protein chemical exchange in membrane-bound proteins is an important parameter for understanding how proteins interact with their aqueous environment, but has been difficult to observe in membrane-bound biological systems. Here, we demonstrate the feasibility of probing specific water-protein chemical exchange in membrane-bound proteins in solid-state MAS NMR. By spin-locking the (1)H magnetization along the magic angle, the (1)H spin diffusion is suppressed such that a water-protein chemical exchange process can be monitored indirectly by dipolar-dephased (15)N signals through polarization transfer from (1)H. In the example of the Influenza A full length M2 protein, the buildup of dipolar-dephased (15)N signals from the tetrad of His37 side chains have been observed as a function of spin-lock time. This confirms that hydronium ions are in exchange with protons in the His37 NH bonds at the heart of the M2 proton conduction mechanism, with an exchange rate constant of ∼1750 s(-1) for pH 6.2 at -10 °C.

  5. 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

    2015-02-05

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. 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. Copyright © 2011 John Wiley & Sons, Ltd.

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

    PubMed

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

    2007-08-23

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

  8. Evidence for the binding of phosphate ion to the C-terminus region in Abeta1-40 using heteronuclear NMR analyses.

    PubMed

    Nagata-Uchiyama, Makiko; Abe, Yoshito; Monji, Akira; Kanba, Shigenobu; Ueda, Tadashi

    2010-02-01

    Amyloid fibril formation of amyloid beta peptide 1-40 (Abeta 1-40) was reported to be retarded in the presence of 150mM phosphate buffer at pH 7 [Monji, Ustumi, Ueda, Imoto, Yoshida, Hashioka, Tashiro and Tashiro, J. Neurochemistry, 77, 1425-1432 (2007)]. In order to elucidate the reason why phosphate ion retards the amyloid fibril formation, we examined the preferential binding sites of phosphate ion to Abeta 1-40 using chemical shift perturbation analysis of heteronuclear NMR. In titration analysis of (15)N-labeled Abeta1-40 in the presence of 150 mM phosphate ion or 150 mM chloride ion, we identified the residues affected by these ions in Abeta 1-40. As a result, we found the tendency that phosphate ion preferentially bound to some residues located on the C-terminus region where the region was reported to be the potential beta-strand region in Abeta1-40. Therefore, we suggested that phosphate ions interacted with the potential beta-strand region in Abeta1-40 to be hard to form beta-sheet in Abeta 1-40, resulting in retardation of the amyloid fibril formation.

  9. Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR.

    PubMed

    Takeda, Mitsuhiro; Sugimori, Nozomi; Torizawa, Takuya; Terauchi, Tsutomu; Ono, Akira M; Yagi, Hirokazu; Yamaguchi, Yoshiki; Kato, Koichi; Ikeya, Teppei; Jee, Jungoo; Güntert, Peter; Aceti, David J; Markley, John L; Kainosho, Masatsune

    2008-12-01

    The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform (13)C/(15)N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally (2)H/(13)C/(15)N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign (1)H, (13)C and (15)N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three beta-sheets.

  10. Contact replacement for NMR resonance assignment.

    PubMed

    Xiong, Fei; Pandurangan, Gopal; Bailey-Kellogg, Chris

    2008-07-01

    Complementing its traditional role in structural studies of proteins, nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in functional studies. NMR dynamics experiments characterize motions involved in target recognition, ligand binding, etc., while NMR chemical shift perturbation experiments identify and localize protein-protein and protein-ligand interactions. The key bottleneck in these studies is to determine the backbone resonance assignment, which allows spectral peaks to be mapped to specific atoms. This article develops a novel approach to address that bottleneck, exploiting an available X-ray structure or homology model to assign the entire backbone from a set of relatively fast and cheap NMR experiments. We formulate contact replacement for resonance assignment as the problem of computing correspondences between a contact graph representing the structure and an NMR graph representing the data; the NMR graph is a significantly corrupted, ambiguous version of the contact graph. We first show that by combining connectivity and amino acid type information, and exploiting the random structure of the noise, one can provably determine unique correspondences in polynomial time with high probability, even in the presence of significant noise (a constant number of noisy edges per vertex). We then detail an efficient randomized algorithm and show that, over a variety of experimental and synthetic datasets, it is robust to typical levels of structural variation (1-2 AA), noise (250-600%) and missings (10-40%). Our algorithm achieves very good overall assignment accuracy, above 80% in alpha-helices, 70% in beta-sheets and 60% in loop regions. Our contact replacement algorithm is implemented in platform-independent Python code. The software can be freely obtained for academic use by request from the authors.

  11. Contact replacement for NMR resonance assignment

    PubMed Central

    Xiong, Fei; Pandurangan, Gopal; Bailey-Kellogg, Chris

    2008-01-01

    Motivation: Complementing its traditional role in structural studies of proteins, nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in functional studies. NMR dynamics experiments characterize motions involved in target recognition, ligand binding, etc., while NMR chemical shift perturbation experiments identify and localize protein–protein and protein–ligand interactions. The key bottleneck in these studies is to determine the backbone resonance assignment, which allows spectral peaks to be mapped to specific atoms. This article develops a novel approach to address that bottleneck, exploiting an available X-ray structure or homology model to assign the entire backbone from a set of relatively fast and cheap NMR experiments. Results: We formulate contact replacement for resonance assignment as the problem of computing correspondences between a contact graph representing the structure and an NMR graph representing the data; the NMR graph is a significantly corrupted, ambiguous version of the contact graph. We first show that by combining connectivity and amino acid type information, and exploiting the random structure of the noise, one can provably determine unique correspondences in polynomial time with high probability, even in the presence of significant noise (a constant number of noisy edges per vertex). We then detail an efficient randomized algorithm and show that, over a variety of experimental and synthetic datasets, it is robust to typical levels of structural variation (1–2 AA), noise (250–600%) and missings (10–40%). Our algorithm achieves very good overall assignment accuracy, above 80% in α-helices, 70% in β-sheets and 60% in loop regions. Availability: Our contact replacement algorithm is implemented in platform-independent Python code. The software can be freely obtained for academic use by request from the authors. Contact: gopal@cs.purdue.edu; cbk@cs.dartmouth.edu PMID:18586716

  12. (S)Pinning down protein interactions by NMR

    PubMed Central

    Kunze, Micha Ben Achim; Erlendsson, Simon

    2017-01-01

    Abstract Protein molecules are highly diverse communication platforms and their interaction repertoire stretches from atoms over small molecules such as sugars and lipids to macromolecules. An important route to understanding molecular communication is to quantitatively describe their interactions. These types of analyses determine the amounts and proportions of individual constituents that participate in a reaction as well as their rates of reactions and their thermodynamics. Although many different methods are available, there is currently no single method able to quantitatively capture and describe all types of protein reactions, which can span orders of magnitudes in affinities, reaction rates, and lifetimes of states. As the more versatile technique, solution NMR spectroscopy offers a remarkable catalogue of methods that can be successfully applied to the quantitative as well as qualitative descriptions of protein interactions. In this review we provide an easy‐access approach to NMR for the non‐NMR specialist and describe how and when solution state NMR spectroscopy is the method of choice for addressing protein ligand interaction. We describe very briefly the theoretical background and illustrate simple protein–ligand interactions as well as typical strategies for measuring binding constants using NMR spectroscopy. Finally, this review provides examples of caveats of the method as well as the options to improve the outcome of an NMR analysis of a protein interaction reaction. PMID:28019676

  13. HDM-PAMPA to predict gastrointestinal absorption, binding percentage, equilibrium and kinetics constants with human serum albumin and using 2 end-point measurements.

    PubMed

    Bujard, Alban; Petit, Charlotte; Carrupt, Pierre-Alain; Rudaz, Serge; Schappler, Julie

    2017-01-15

    The parallel artificial membrane permeability assay (PAMPA) is a high-throughput screening (HTS) technique developed to predict passive permeability through numerous different biological membranes, such as the gastrointestinal tract (GIT), the blood brain barrier (BBB), and the dermal layer. PAMPA is based on an artificial membrane, such as hexadecane (HDM), which separates two compartments (i.e., a donor and an acceptor compartment). In the present study, an HDM-PAMPA method was developed with human serum albumin (HSA) under iso-pH and gradient-pH conditions to predict the percentage of binding, dissociation/association constants (Kd and Ka, respectively) and dissociation/association kinetic rates (koff and kon, respectively) between a given drug and HSA. Thanks to the kinetic properties of PAMPA, a two end-point assay was implemented to obtain all three properties. The assay was used to measure basic, acidic, and amphoteric compounds. The protein was free in solution, allowing a direct comparison between this assay and equilibrium dialysis (ED). The developed PAMPA enabled screening of up to 96 compounds in a single run, generating valuable information on absorption and distribution in a high-throughput and high-repeatable manner.

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

    PubMed

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

    2005-11-01

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

  15. 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.

  16. 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

  17. The prion protein binds thiamine.

    PubMed

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

    2011-11-01

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

  18. 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.

  19. 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.

  20. Revision of the affinity constant for perchlorate binding to the sodium-iodide symporter based on in vitro and human in vivo data.

    PubMed

    Schlosser, Paul M

    2016-12-01

    A series of previously published physiologically based pharmacokinetic (PBPK) models describe the effect of perchlorate on iodide uptake by the thyroid, with the mechanism being competitive inhibition of iodide transport by the sodium-iodide symporter (NIS). Hence a key parameter of these models is the affinity of perchlorate for the NIS, characterized as the Michaelis-Menten kinetic constant, Km . However, when model predictions were compared to published results of a human study measuring radio-iodide uptake (RAIU) inhibition after controlled perchlorate exposures, it was found to only fit the lowest exposure level and underpredicted RAIU inhibition at higher levels. Published in vitro data, in which perchlorate-induced inhibition of iodide uptake via the NIS was measured, were re-analyzed. Km for binding of perchlorate to the NIS originally derived from these data, 1.5 μm, had been obtained using Lineweaver-Burk plots, which allow for linear regression but invert the signal-noise of the data. Re-fitting these data by non-linear regression of the non-inverted data yielded a 60% lower value for the Km , 0.59 μm. Substituting this value into the PBPK model for an average adult human significantly improved model agreement with the human RAIU data for exposures <100 μg kg(-1)  day(-1) . Thus, this lower Km value both fits the in vitro NIS kinetics and provides better predictions of human in vivo RAIU data. This change in Km increases the predicted sensitivity of humans to perchlorate over twofold for low-level exposures. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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

    DTIC Science & Technology

    1989-05-24

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

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

    PubMed

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

    2011-12-02

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

  3. Monte Carlo simulations of the peptide recognition at the consensus binding site of the constant fragment of human immunoglobulin G: the energy landscape analysis of a hot spot at the intermolecular interface.

    PubMed

    Verkhivker, Gennady M; Bouzida, Djamal; Gehlhaar, Daniel K; Rejto, Paul A; Freer, Stephan T; Rose, Peter W

    2002-08-15

    Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13-residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium-simulated tempering dynamics with the simplified, knowledge-based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met-252, Ile-253, His-433, and His-435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn-434, Ser-254, and Tyr-436 protein residues contribute significantly to the binding affinity of the peptide-protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex.

  4. Binding site of Zn 2+ in ATP:N1 at low pH and N7 at high pH as evidenced by 1H 15N NMR HMBC experiments

    NASA Astrophysics Data System (ADS)

    Du, Fei; Mao, Xi-An

    2000-11-01

    Nuclear magnetic resonance (NMR) 1H 15N heteronuclear multiple bond correlation (HMBC) experiments on natural abundant adenosine-5'-triphosphate (ATP) showed that zinc(II) induced large chemical shifts (>10 ppm) for N1 at lower pH (2-5) while for N7 at higher pH (5-7), suggesting that the binding site of Zn 2+ in the purine base of ATP is pH dependent. The size effect of zinc(II) in coordination is also discussed.

  5. Calorimetric investigation of proton linkage by monitoring both the enthalpy and association constant of binding: application to the interaction of the Src SH2 domain with a high-affinity tyrosyl phosphopeptide.

    PubMed

    Bradshaw, J M; Waksman, G

    1998-11-03

    The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have used isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's association constant between pH 5.0 and 9.0, two ionizable groups were characterized. One group, with free and bound pKas of 6.2 and 4.4, respectively, could be identified as the phosphate in the phosphotyrosine while the other group, with free and bound pKas of 8.2 and 8.5, respectively, could be only tentatively assigned to a cysteine in the phosphotyrosine binding pocket. Further information on the linkage between peptide binding and protonation of the phosphotyrosine was obtained from a second series of experiments, which focused on determining the peptide binding enthalpy at low values of pH in several buffers with different ionization enthalpies. These data provided free and bound pKa values for the phosphotyrosine identical to those derived from the first series of experiments, and hence demonstrated for the first time that the two approaches provide identical information regarding proton linkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotonated phosphate forms of the peptide. These two sets of experiments provided a complete energetic profile of the linkage between phosphate ionization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol-1 more favorably than the PO3H1- form due entirely to a more favorable entropy of binding.

  6. 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…

  7. 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…

  8. 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)

  9. 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)

  10. Solution-state (15)N NMR spectroscopic study of alpha-C-phycocyanin: implications for the structure of the chromophore-binding pocket of the cyanobacterial phytochrome Cph1.

    PubMed

    Hahn, Janina; Kühne, Ronald; Schmieder, Peter

    2007-12-17

    The detailed structure of the chromophore-binding pocket in phytochrome proteins and the structural changes associated with its photocycle are still matters of debate. Insight into the structure and dynamics of the binding pocket has been gained through the comparison of a (15)N NMR spectrum of alpha-C-phycocyanin, which is often used as a model system for the study of phytochromes, with the previously described (15)N NMR spectrum of the cyanobacterial phytochrome Cph1. The former spectrum supports the hypothesis that all four nitrogen atoms of the alpha-C-phycocyanin chromophore are protonated, in analogy with the proposed protonation state for the P(r) and P(fr) forms of Cph1. The spectra show that the chromophores in both proteins exhibit a distinct dynamic behavior, as also indicated by a NOESY spectrum of Cph1. Finally, stereochemical arguments and a Cph1 homology model support the hypothesis that the chromophore in Cph1 is most likely in the ZZZssa conformation in the P(r) form of the protein.

  11. 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.

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

    PubMed

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

    2008-08-06

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

  13. Transient state kinetics of enzyme IICBGlc, a glucose transporter of the phosphoenolpyruvate phosphotransferase system of Escherichia coli: equilibrium and second order rate constants for the glucose binding and phosphotransfer reactions.

    PubMed

    Meadow, Norman D; Savtchenko, Regina S; Nezami, Azin; Roseman, Saul

    2005-12-23

    During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA(Glc) and Enzyme IICB(Glc), the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA(Glc) to IICB(Glc) to Glc) and also the second order rate constants for the transfer from phospho-IIA(Glc) to molecularly cloned IIB(Glc), the soluble, cytoplasmic domain of IICB(Glc). The rate constants for the forward and reverse phosphotransfer reactions between IIA(Glc) and IICB(Glc) were 3.9 x 10(6) and 0.31 x 10(6) m(-1) s(-1), respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB(Glc) and Glc was 3.2 x 10(6) m(-1) s(-1). From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIA(Glc) to the phosphorylation site Cys of IIB(Glc) or IICB(Glc) were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB(Glc) were invariably found to contain endogenous, firmly bound Glc (estimated K'(D) approximately 10(-7) m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB(Glc) has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.

  14. Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin.

    PubMed

    Zanotti, J M; Bellissent-Funel, M C; Parello, J

    1999-05-01

    The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.

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

    PubMed Central

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

    2013-01-01

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

  16. Binding of perfluorooctanoic acid to rat and human plasma proteins.

    PubMed

    Han, Xing; Snow, Timothy A; Kemper, Raymond A; Jepson, Gary W

    2003-06-01

    Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. (19)F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be approximately 0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA and PFOA binding to HSA. The dissociation constants for binding of PFOA to RSA or HSA and the numbers of PFOA binding sites were in the range of 0.3-0.4 mM and 6-9, respectively. On the basis of these binding parameters and the estimated plasma concentration of serum albumin, greater than 90% of PFOA would be bound to serum albumin in both rat and human blood.

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

    PubMed

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

    2008-10-21

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

  18. 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

  19. A theoretical study of the NMR spin-spin coupling constants of the complexes [(NC)(5)Pt-Tl(CN)(n)](n-) (n = 0-3) and [(NC)(5)Pt-Tl-Pt(CN)(5)](3-): a lesson on environmental effects.

    PubMed

    Autschbach, Jochen; Le Guennic, Boris

    2003-11-05

    The molecular geometries and the nuclear spin-spin coupling constants of the complexes [(NC)(5)Pt-Tl(CN)(n)](n-), n = 0-3, and the related system [(NC)(5)Pt-Tl-Pt(CN)(5)](3-) are studied. These complexes have received considerable interest since the first characterization of the n = 1 system by Glaser and co-workers in 1995 [J. Am. Chem. Soc. 1995, 117, 7550-7551]. For instance, these systems exhibit outstanding NMR properties, such as extremely large Pt-Tl spin-spin coupling constants. For the present work, all nuclear spin-spin coupling constants J(Pt-Tl), J(Pt-C), and J(Tl-C) have been computed by means of a two-component relativistic density functional approach. It is demonstrated by the application of increasingly accurate computational models that both the huge J(Pt-Tl) for the complex (NC)(5)Pt-Tl and the whole experimental trend among the series are entirely due to solvent effects. An approximate inclusion of the bulk solvent effects by means of a continuum model, in addition to the direct coordination, proves to be crucial. Similarly drastic effects are reported for the coupling constants between the heavy atoms and the carbon nuclei. A computational model employing the statistical average of orbital-dependent model potentials (SAOP) in addition to the solvent effects allows to accurately reproduce the experimental coupling constants within reasonable limits.

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

    PubMed

    Li, Xiang; Shantz, Daniel F

    2011-04-05

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

  1. Simultaneously cycled NMR spectroscopy.

    PubMed

    Parish, David M; Szyperski, Thomas

    2008-04-09

    Simultaneously cycled (SC) NMR was introduced and exemplified by implementing a set of 2-D [1H,1H] SC exclusive COSY (E.COSY) NMR experiments, that is, rf pulse flip-angle cycled (SFC), rf pulse phase cycled (SPC), and pulsed field gradient (PFG) strength cycled (SGC) E.COSY. Spatially selective 1H rf pulses were applied as composite pulses such that all steps of the respective cycles were affected simultaneously in different slices of the sample. This increased the data acquisition speed for an n-step cycle n-fold. A high intrinsic sensitivity was achieved by defining the cycles in a manner that the receiver phase remains constant for all steps of the cycle. Then, the signal resulting from applying the cycle corresponded to the sum of the signals from all steps of the cycle. Hence, the detected free induction decay did not have to be separated into the contributions arising from different slices, and read-out PFGs, which not only greatly reduce sensitivity but also negatively impact lineshapes in the direct dimension, were avoided. The current implementation of SFC E.COSY reached approximately 65% of the intrinsic sensitivity of the conventional phase cycled congener, making this experiment highly attractive whenever conventional data acquisition is sampling limited. Highly resolved SC E.COSY yielding accurate 3J-coupling values was recorded for the 416 Da plant alkaloid tomatidine within 80 min, that is, 12 times faster than with conventional phase cycled E.COSY. SC NMR is applicable for a large variety of NMR experiments and thus promises to be a valuable addition to the arsenal of approaches for tackling the NMR sampling problem to avoid sampling limited data acquisition.

  2. The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies1[OPEN

    PubMed Central

    Wang, Tuo; Hong, Mei

    2016-01-01

    The wall-loosening actions of β-expansins are known primarily from studies of EXPB1 extracted from maize (Zea mays) pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state nuclear magnetic resonance experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing 13C-enriched maize CWs with EXPB1 functionalized with a Mn2+ tag, we measured Mn2+-induced PRE. Strong 1H and 13C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX. In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly substituted GAX and GAX of low substitution, which binds cellulose. This study provides molecular evidence of β-expansin’s target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously. PMID:27729469

  3. Structural and dynamic aspects of binding of prototype lexitropsin to the decadeoxyribonucleotide d(CGCAATTGCC)/sub 2/ deduced from high-resolution /sup 1/H NMR studies

    SciTech Connect

    Lee, M.; Chang, D.K.; Hartley, J.A.; Pon, R.T.; Krowicki, K.; Lown, J.W.

    1988-01-12

    Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d-(CGCAATTGCC)/sub 2/ and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide and are deduced by using high-resolution /sup 1/H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)/sub 2/ have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the /sup 1/H NMR spectrum of the l:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH/sub 3/(6), and T-CH/sub 3/(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Strong nuclear Overhauser effects (NOEs) between pyrrol H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Alternative mechanisms for this exchange process are considered.

  4. Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies.

    PubMed

    Sharma, Alok K; Sawhney, Paramvir; Memisoglu, Gonen; Rigby, Alan C

    2012-01-01

    Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.

  5. The binding of cytochrome c peroxidase and ferricytochrome c. A spectrophotometric determination of the equilibrium association constant as a function of ionic strength.

    PubMed

    Erman, J E; Vitello, L B

    1980-07-10

    Complex formation between cytochrome c peroxidase and ferricytochrome c perturbs the optical absorption spectrum in the Soret band by about 2%. This perturbation can be utilized as a measure of the complex formed in solution and permits the determination of the stoichiometry and the equilibrium association constant for this reaction. At pH 6, in cacodylate/KNO3 buffers, only a 1:1 complex between cytochrome c peroxidase and ferricytochrome c is detected. The equilibrium association constant for the complex has been determined as a function of ionic strength and varies between (6.0 +/- 3.6) x 10(6) M-1 and (2.2 +/- 1.9) x 10(6) M-1 over the ionic strength range 0.01 M to 0.20 M.

  6. 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

  7. Macrocycles Containing Tin. 119Sn NMR Studies of Chloride Binding by Lewis Acidic Tin Compounds. Multidentiate Effects, Macrocyclic Effects and Size Selectivity.

    DTIC Science & Technology

    1985-09-15

    complexation of 1-4 and dibutyltin dichloride (5) with chloride ion in acetonitrile was studied by Sn-119 NMR spectroscopy. Rapid exchange of chloride between...been prepared as has an acyclic di-tin model (5,5,16,16- *. tetrachloro-5,16-distannadocosane, 4). The complexation of 1-4 and dibutyltin dichloride (5...I or 3. Thus, for comparison, acyclic *model compound 4 was prepared by the sequence shown in Scheme 2. Dibutyltin dichloride (5) was used as a model

  8. 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)

  9. 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)

  10. (133)Cs NMR and molecular dynamics simulation on bilayers of Cs(+) ion binding to aggregates of fatty acid soap at high pH.

    PubMed

    Xu, Wenlong; Zhang, Heng; Dong, Shuli; Hao, Jingcheng

    2014-10-07

    Fatty acid bilayers are usually formed due to the hydrogen bonds between the protonated carboxyl (-COOH) and the deprotonated carboxylate (-COO(-)). Therefore, the formation of the bilayers must be at the pH around the pKa of the fatty acid, which is a narrow pH range (mostly about 7-9). Fatty acid bilayers can be used as cell membrane model but the narrow pH range largely limits their applications. Herein, fatty acid bilayers were first detected at high pH (>13) in the stearic acid (SA)/CsOH/H2O system, which is not consistent with the explanation of the traditional hydrogen bond theory for fatty acid bilayers around pH. Cryogenic transmission electron microscopy (cryo-TEM) images, X-ray diffraction (XRD) patterns, and deuterium nuclear magnetic resonance ((2)H NMR) spectra demonstrate the planar sheet bilayers. The pH, conductivity, and (133)Cs NMR data indicate the strong interaction between Cs(+) and the bilayers. Rheological characterizations reflect the viscoelasticity of the Lα phase sample of bilayers. Molecular dynamics simulation increases the reliability of our observations. The assumed growth process of the aggregates and the detailed arrangement of the Cs(+) on the bilayers were proposed according to the experimental data and the molecular dynamics simulation. This work will promote the application scope of fatty acid bilayers with wide pH range.

  11. Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy.

    PubMed

    Yuasa, Noriyuki; Koyama, Tsubasa; Subedi, Ganesh P; Yamaguchi, Yoshiki; Matsushita, Misao; Fujita-Yamaguchi, Yoko

    2013-12-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

  12. Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean.

    PubMed

    Battistuzzi, G; Dietrich, M; Löcke, R; Witzel, H

    1997-05-01

    The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

  13. Measurement of the Dissociation-Equilibrium Constants for Low Affinity Antibiotic Binding Interaction with Bacterial Ribosomes by the T2 (CPMG) and Line-Broadening Methods

    NASA Astrophysics Data System (ADS)

    Verdier, L.; Gharbi-Benarous, J.; Bertho, G.; Mauvais, P.; Girault, J.-P.

    1999-10-01

    In this study the dissociation constants of the low antibiotic-ribosomes interaction were determined by the T2 (CPMG), the Carr-Purcell-Meiboom-Gill spin-echo decay rate and the line-broadening methods. Three MLSB antibiotics were studied, a macrolide roxithromycin, a ketolide HMR 3647 and a lincosamide clindamycin for their weak interaction with three bacterial ribosomes, E. coli, Staphylococcus aureus sensitive and resistant to erythromycin. Nous avons mesuré la constante de dissociation, Kd correspondant à l'interaction faible antibiotique-ribosome bactérien pour des antibiotiques de différentes classes, un macrolide (roxithromycine), un kétolide (HMR 3647) et une lincosamide (clindamycine) avec des ribosomes de différentes souches bactériennes (E. coli, Staphylococcus aureus sensible ou résistant à l'erythromycin) par deux méthodes : l'une basée sur la variation des largeurs de raies et l'autre sur les temps de relaxation transversaux T2 en utilisant une séquence CPMG.

  14. Multinuclear NMR and molecular modelling investigations on the structure and equilibria of complexes that form in aqueous solutions of Ca(2+) and gluconate.

    PubMed

    Pallagi, Attila; Sebok, Pál; Forgó, Péter; Jakusch, Tamás; Pálinkó, István; Sipos, Pál

    2010-09-03

    Complexation of d-gluconate (Gluc(-)) with Ca(2+) has been investigated via (1)H, (13)C and (43)Ca NMR spectroscopy in aqueous solutions in the presence of high concentration background electrolytes (1MI4M (NaCl) ionic strength). From the ionic strength dependence of its formation constant, the stability constant at 6pH11 and at I-->0M has been derived (logK(1,1)(0)=1.8+/-0.1). The protonation constant of Gluc(-) at I=1M (NaCl) ionic strength was also determined and was found to be logK(a)=3.24+/-0.01 ((13)C NMR) and logK(a)=3.23+/-0.01 ((1)H NMR). It was found that (1)H and (13)C NMR chemical shifts upon complexation (both with H(+) and with Ca(2+)) do not vary in an unchanging way with the distance from the Ca(2+)/H(+) binding site. From 2D (1)H-(43)Ca NMR spectra, simultaneous binding of Ca(2+) to the alcoholic OH on C2 and C3 was deduced. Molecular modelling results modulated this picture by revealing structures in which the Gluc(-) behaves as a multidentate ligand. The five-membered chelated initial structure was found to be thermodynamically more stable than that derived from a six-membered chelated initial structure.

  15. 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

  16. Use of capillary electrophoresis as a versatile tool to measure interaction constants between a KDR-binding PEGylated lipopeptide and pegylated phospholipid micelles.

    PubMed

    Poitevin, Martine; Tranquart, François; Cherkaoui, Samir

    2015-01-01

    In the frame of our molecular imaging activities, a PEGylated lipopeptide has been developed as a specific ligand for the human vascular endothelial growth factor receptor 2, which is considered as one of the important molecular marker of angiogenesis. In this study, the potential of affinity capillary electrophoresis (ACE) is evaluated to measure the interactions of an active PEGylated lipopeptide, its hydrolysis product and its precursor consisting of a peptide structure with different micelles including Brij-35, Tween-20, and pegylated phospholipids. Given the amphiphilic structure of the PEGylated lipopeptide, a MEKC method allowing the simultaneous separation of the compounds of interest was set up, using low percentages of acetonitrile. Analytes were resolved using a BGE consisting of 100 mM borate buffer pH 9.0, 1 mM Brij, and 25% acetonitrile. Optimized conditions were then used to perform ACE experiments. The affinity constants of the analytes with the micelles were calculated on the basis of their mobility decrease when surfactant concentration increased in the electrolyte. The use of different linearization models to estimate affinity constants was discussed and comparison of different surfactants was reported. PEGylated lipopeptide interacted more strongly with pegylated phospholipid micelles than with Brij-35 or Tween-20. Moreover, it is likely that the chemical structure of the compounds, and particularly the lipidic part of the molecules, significantly affects the interaction with micelles. In conclusion, the ACE method can be readily applied to investigate interactions of our targeting lipopeptides with various micelles currently used for the preparation of pharmaceutical vehicles.

  17. 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.

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

    PubMed

    Casares, Salvador; Ab, Eiso; Eshuis, Henk; Lopez-Mayorga, Obdulio; van Nuland, Nico A J; Conejero-Lara, Francisco

    2007-04-02

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

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

    PubMed Central

    Casares, Salvador; AB, Eiso; Eshuis, Henk; Lopez-Mayorga, Obdulio; van Nuland, Nico AJ; Conejero-Lara, Francisco

    2007-01-01

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

  20. Nuclear magnetic resonance (NMR) solution structure, dynamics, and binding properties of the kringle IV type 8 module of apolipoprotein(a).

    PubMed

    Chitayat, Seth; Kanelis, Voula; Koschinsky, Marlys L; Smith, Steven P

    2007-02-20

    The plasma lipoprotein lipoprotein(a) [Lp(a)] comprises a low-density lipoprotein (LDL)-like particle covalently attached to the glycoprotein apolipoprotein(a) [apo(a)]. Apo(a) consists of multiple tandem repeating kringle modules, similar to plasminogen kringle IV (designated KIV1-KIV10), followed by modules homologous to the kringle V module and protease domain of plasminogen. The apo(a) KIV modules have been classified on the basis of their binding affinity for lysine and lysine analogues. The strong lysine-binding apo(a) KIV10 module mediates lysine-dependent interactions with fibrin and cell-surface receptors. Weak lysine-binding apo(a) KIV7 and KIV8 modules display a 2-3-fold difference in lysine affinity and play a direct role in the noncovalent step in Lp(a) assembly through binding to unique lysine-containing sequences in apolipoproteinB-100 (apoB-100). The present study describes the nuclear magnetic resonance solution structure of apo(a) KIV8 and its solution dynamics properties, the first for an apo(a) kringle module, and compares the effects of epsilon-aminocaproic acid (epsilon-ACA) binding on the backbone and side-chain conformation of KIV7 and KIV8 on a per residue basis. Apo(a) KIV8 adopts a well-ordered structure that shares the general tri-loop kringle topology with apo(a) KIV6, KIV7, and KIV10. Mapping of epsilon-ACA-induced chemical-shift changes on KIV7 and KIV8 indicate that the same residues are affected, despite a 2-3-fold difference in epsilon-ACA affinity. A unique loop conformation within KIV8, involving hydrophobic interactions with Tyr40, affects the positioning of Arg35 relative to the lysine-binding site (LBS). A difference in the orientation of the aromatic side chains comprising the hydrophobic center of the LBS in KIV8 decreases the size of the hydrophobic cleft compared to other apo(a) KIV modules. An exposed hydrophobic patch contiguous with the LBS in KIV8 and not conserved in other weak lysine-binding apo(a) kringle modules

  1. Influence of Mg2+ and pH on n.m.r. spectra and radioligand binding of inositol 1,4,5-trisphosphate.

    PubMed Central

    White, A M; Varney, M A; Watson, S P; Rigby, S; Liu, C S; Ward, J G; Reese, C B; Graham, H C; Williams, R J

    1991-01-01

    We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3. PMID:1654891

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

    PubMed

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

    2015-01-08

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

  3. Size and alloying induced changes in lattice constant, core, and valance band binding energy in Pd-Ag, Pd, and Ag nanoparticles: Effect of in-flight sintering temperature

    NASA Astrophysics Data System (ADS)

    Sengar, Saurabh K.; Mehta, B. R.; Govind

    2012-07-01

    In the present study, we report the growth of size selected Pd, Ag, and Pd-Ag alloy nanoparticles by an integrated method comprising of the gas phase synthesis, electrical mobility size selection, and in-flight sintering steps. Effect of temperature during in-flight sintering on nanoparticle size, crystal structure, and electronic properties has been studied. XRD studies show lattice expansion in Pd and Ag nanoparticles and lattice contraction in Pd-Ag alloy nanoparticles on increasing the sintering temperatures. In case of Pd and Ag nanoparticles, size induced changes in lattice constants are consistent with the changes in the binding energy positions with respect to bulk values. In case of Pd-Ag alloy nanoparticles, change in nanoparticle size and composition on sintering affect the lattice constant and binding energy positions. Large changes in Pd4d valance band centroid in Pd-Ag nanoparticles are due to size and alloying effects. The results of this study are important for understanding the correlation between electronic properties and Pd-H interaction in Pd alloy nanoparticles.

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

    PubMed

    Sauvé, Simon; Tremblay, Luc; Lavigne, Pierre

    2004-09-17

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

  5. Structure and stability of a DNA triple helix in solution: NMR studies on d(T) sub 6 ter dot d(T) sub 6 and its complex with a minor groove binding drug

    SciTech Connect

    Umemoto, Kimiko; Sarma, Mukti H.; Gupta, Goutam; Luo, Jia; Sarma, Ramaswamy H. )

    1990-05-23

    The possibility of both Watson-Crick and Hoogsteen A{center dot}T pairs can result in a triple helical structure for d(T){sub 6}{center dot}d(A){sub 6}{center dot}d(T){sub 6} in solution. In the triple helix the Watson-Crick paired T strand can run antiparallel, while the Hoogsteen paired T strand can run parallel to the A strand. On the basis of 1D/2D NMR studies, we have characterized the structural properties of the triple helix in terms of (a) nature of H-bonding, (b) chain conformations and relative chain orientations, (c) location of triplets T{center dot}A{center dot}T with respect to the helix axis, and (d) effects of NaCl and MgCl{sub 2}. In addition, we experimentally demonstrate that a minor groove specific drug Dst2 (a distamycin analogue) can bind to the triple helix. We show that the nature of thermal transition is altered by Dst2 binding; i.e., the host triple helix shows triple {yields} coil (monophasic) transition in the absence of Dst2, while in its presence the helix shows a triplex {yields} duplex {yields} coil (biphasic) transition.

  6. 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

  7. Lanthanide Ions Bind Specifically to an Added ``EF-Hand'' and Orient a Membrane Protein in Micelles for Solution NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Ma, Che; Opella, Stanley J.

    2000-10-01

    Twelve amino acid residues corresponding to an "EF-hand" calcium-binding site were added to the N-terminus of a protein, providing a specific lanthanide ion binding that weakly orients the protein in solution. A comparison of spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modification, since there are minimal chemical shift changes. With a lanthanide but not calcium bound to the EF-hand, the protein is weakly oriented by the magnetic field, since residual dipolar couplings can be measured. Since the signs and magnitudes of the couplings varied with the type of lanthanide, this demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrupted the commonly employed bicelle and filamentous phage solutions; therefore, the addition of a specific metal binding site in the form of an EF-hand may provide a general approach to orienting proteins where the addition of external agents is problematic. An additional benefit is that the lanthanide ions perturb the protein resonances in ways that provide unique orientational and distance constraints.

  8. 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. Copyright © 2014. Published by Elsevier Inc.

  9. 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

  10. 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.

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

    PubMed

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

    2011-01-20

    An integrated computational methodology for interpreting NMR spin relaxation in proteins has been developed. It combines a two-body coupled-rotator stochastic model with a hydrodynamics-based approach for protein diffusion, together with molecular dynamics based calculations for the evaluation of the coupling potential of mean force. The method is applied to ¹⁵N relaxation of N-H bonds in the Rho GTPase binding (RBD) domain of plexin-B1, which exhibits intricate internal mobility. Bond vector dynamics are characterized by a rhombic local ordering tensor, S, with principal values S₀² and S₂², and an axial local diffusion tensor, D₂, with principal values D(2,||) and D(2,⊥). For α-helices and β-sheets we find that S₀² ~ -0.5 (strong local ordering), -1.2 < S₂² < -0.8 (large S tensor anisotropy), D(2,⊥) ~ D₁ = 1.93 × 10⁷ s⁻¹ (D₁ is the global diffusion rate), and log(D(2,||)/D₁) ~ 4. For α-helices the z-axis of the local ordering frame is parallel to the C(α)-C(α) axis. For β-sheets the z-axes of the S and D₂ tensors are parallel to the N-H bond. For loops and terminal chain segments the local ordering is generally weaker and more isotropic. On average, D(2,⊥) ~ D₁ also, but log(D(2,||)/D₁) is on the order of 1-2. The tensor orientations are diversified. This study sets forth an integrated computational approach for treating NMR relaxation in proteins by combining stochastic modeling and molecular dynamics. The approach developed provides new insights by its application to a protein that experiences complex dynamics.

  12. 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.

  13. Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: the Role of Ser31 in Amantadine Binding

    PubMed Central

    Cady, Sarah D.; Mishanina, Tatiana V.; Hong, Mei

    2014-01-01

    The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the sidechain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting

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

    PubMed

    Ghiladi, Reza A; Karlin, Kenneth D

    2002-05-06

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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. A two-dimensional NMR study of the solution structure of a DNA dodecamer comprising the concensus sequence for the specific DNA-binding sites of the glucocorticoid receptor protein.

    PubMed

    Clore, G M; Lauble, H; Frenkiel, T A; Gronenborn, A M

    1984-12-17

    A two-dimensional 500-MHz 1H-NMR study on the non-self-complementary double-stranded DNA dodecamer 5'd(C-C-A-G-A-A-C-A-G-T-G-G)5'd(C-C-A-C-T-G-T-T-C-T-G-G), is presented. This oligonucleotide contains the consensus octanucleotide sequence 5'd(A-G-A-A-C-A-G-T) for the specific DNA-binding sites of the glucocorticoid receptor protein [Payvar, F. et al. (1984) Cell 35, 381-392]. Using a combination of two-dimensional pure phase absorption nuclear Overhauser enhancement (NOESY) and homonuclear J-correlated (COSY) spectroscopy all non-exchangeable base (with the exception fo the adenine H2 protons), methyl and deoxyribose H1', H2', H2", H3' and H4' resonances are assigned unambiguously using a sequential resonance assignment strategy. From the relative intensities of the cross peaks in the pure phase absorption NOESY spectra at two mixing times it is shown that the dodecamer adopts a B-type conformation in solution.

  20. Protein Motions and Folding Investigated by NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Palmer, Arthur

    2002-03-01

    NMR spin relaxation spectroscopy is a powerful experimental approach for globally characterizing conformational dynamics of proteins in solution. Laboratory frame relaxation measurements are sensitive to overall rotational diffusion and internal motions on picosecond-nanosecond time scales, while rotating frame relaxation measurements are sensitive to chemical exchange processes on microsecond-millisecond time scales. The former approach is illustrated by ^15N laboratory-frame relaxation experiments as a function of temperature for the helical subdomain HP36 of the F-actin-binding headpiece domain of chicken villin. The data are analyzed using the model-free formalism to characterize order parameters and effective correlation times for intramolecular motions of individual ^15N sites. The latter approach is illustrated by ^13C Carr-Purcell-Meiboom-Gill relaxation measurements for the de novo designed α_2D protein and by ^15N rotating-frame relaxation measurements for the peripheral subunit-binding domain (PSBD) from the dihydrolopoamide acetyltransferase component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus. These experiments are used to determine the folding and unfolding kinetic rate constants for the two proteins. The results for HP36, α_2D, and PSBD illustrate the capability of current NMR methods for characterizing dynamic processes on multiple time scales in proteins.

  1. 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.

  2. A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL Apical Domain with implications for substrate interactions

    SciTech Connect

    Hua, Q. X. H.; Dementieva, I. S. D.; Walsh, M. A. W.; Hallenga, K. H.; Weiss, M. A. W.; Joachimiak, A. J.; Biosciences Division; Case Western Reserve Univ.; IRBM P. Angeletti; Purdue Univ.

    2001-02-23

    A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (T{sub m}>100{sup o}C as evaluated by circular dichroism) is at least 35{sup o}C greater than that of the E. coli apical domain (T{sub m}=65{sup o}C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 {angstrom} using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 {angstrom} based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the 'Anfinsen cage' in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using {sup 15}N-{sup 1}H correlation spectra of {sup 15}N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50{sup o}C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal {alpha}-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the 'classical' peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.

  3. 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.

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

    SciTech Connect

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

    1998-08-15

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

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