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Sample records for fast kinetic ligands

  1. Dual-point competition association assay: a fast and high-throughput kinetic screening method for assessing ligand-receptor binding kinetics.

    PubMed

    Guo, Dong; van Dorp, Erika J H; Mulder-Krieger, Thea; van Veldhoven, Jacobus P D; Brussee, Johannes; Ijzerman, Adriaan P; Heitman, Laura H

    2013-03-01

    The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A(1) receptor (A(1)R) was chosen for assay validation and optimization. A screen with 35 high-affinity A(1)R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families.

  2. Kinetics of ligand binding to nucleic acids.

    PubMed

    Arakelyan, V B; Babayan, S Y; Tairyan, V I; Arakelyan, A V; Parsadanyan, M A; Vardevanyan, P O

    2006-02-01

    Ligand binding to nucleic acids (NA) is considered as a stationary Markov process. It is shown that the probabilistic description of ligand-NA binding allows one to describe not only the kinetics of the change of number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. The general analysis of absorption isotherms and kinetics of ligand binding to NA make it possible to determine of rate constants of ligand-NA complex formation and dissociation.

  3. Fast protein folding kinetics.

    PubMed

    Gelman, Hannah; Gruebele, Martin

    2014-05-01

    Fast-folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast-folding proteins has provided insight into the mechanisms, which allow some proteins to find their native conformation well <1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even 'slow' folding processes: fast folders are small; relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast-folding proteins and provides an overview of the major findings of fast-folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general, as well as some work that is left to do.

  4. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  5. Adsorption and Exchange Kinetics of Hydrophilic and Hydrophobic Phosphorus Ligands on Gold Surface

    NASA Astrophysics Data System (ADS)

    Zhuge, X. Q.; Bian, Z. C.; Luo, Z. H.; Mu, Y. Y.; Luo, K.

    2017-02-01

    The adsorption kinetics process of hydrophobic ligand (triphenylphosphine, PPh3) and hydrophilic ligand (tris(hydroxymethyl)phosphine oxide, THPO) on the surface of gold electrode were estimated by using electrical double layer capacitance (EDLC). Results showed that the adsorption process of both ligands included fast and slow adsorption processes, and the fast adsorption process could fit the first order kinetic equation of Langmuir adsorption isotherm. During the slow adsorption process, the surface coverage (θ) of PPh3 was higher than that of THPO due to the larger adsorption kinetic constant of PPh3 than that of THPO, which implied that PPh3 could replace THPO on the gold electrode. The exchange process of both ligands on the surface of gold electrode proved that PPh3 take the place of THPO by testing the variation of EDLC which promote the preparation of Janus gold, and the theoretic simulation explained the reason of ligands exchange from the respect of energy..

  6. [Kinetics of ligand binding to nucleic acids at random fillings].

    PubMed

    Arakelian, V B; Babaian, S Iu; Tairian, V I; Arakelian, A V; Parsadanian, M A; Vardevanian, P O

    2006-01-01

    Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

  7. A PET imaging agent with fast kinetics: synthesis and in vivo evaluation of the serotonin transporter ligand [11C]2-[2-dimethylaminomethylphenylthio)]-5-fluorophenylamine ([11C]AFA).

    PubMed

    Huang, Yiyun; Narendran, Raj; Bae, Sung-A; Erritzoe, David; Guo, Ningning; Zhu, Zhihong; Hwang, Dah-Ren; Laruelle, Marc

    2004-08-01

    A new serotonin transporter (SERT) ligand, [11C]2-[2-(dimethylaminomethylphenylthio)]-5-fluorophenylamine (10, [11C]AFA), was synthesized and evaluated as a candidate PET radioligand in pharmacological and pharmacokinetic studies. As a PET radioligand, AFA (8) can be labeled with either C-11 or F-18. In vitro, AFA displayed high affinity for SERT (Ki 1.46 +/- 0.15 nM) and lower affinity for norepinephrine transporter (NET, Ki 141.7 +/- 47.4 nM) or dopamine transporter (DAT, Ki > 10,000 nM). [11C]AFA (10) was prepared from its monomethylamino precursor 9 by reaction with high specific activity [11C]methyl iodide. Radiochemical yield was 43 +/- 20% based on [11C]methyl iodide at end of bombardment (EOB, n = 10) and specific activity was 2,129 +/- 1,369 Ci/mmol at end of synthesis (EOS, n = 10). Biodistribution studies in rats indicated that [11C]AFA accumulated in brain regions known to contain high concentrations of SERT. Binding in SERT-rich brain regions was reduced significantly by pretreatment with either the cold compound 8 or with the selective serotonin reuptake inhibitor (SSRI) citalopram, but not by the selective norepinephrine reuptake inhibitor nisoxetine, thus underlining its in vivo binding selectivity and specificity for SERT. Imaging experiments in baboons demonstrated that the uptake pattern of [11C]AFA in the baboon brain is consistent with the known distribution of SERT, with highest activity levels in the midbrain and thalamus, followed by striatum, hippocampus, and cortical regions. Activity levels in the baboon brain peaked at 15-40 min after radioligand injection, indicating a fast uptake kinetics for [11C]AFA. Pretreatment of the baboon with citalopram (4 mg/kg) significantly reduced the specific binding of [11C]AFA in all SERT-containing brain regions. Kinetic analysis revealed that the regional equilibrium specific to non-specific partition coefficients (V3") of [11C]AFA are similar to those of [11C]McN5652, but lower than those of [11C

  8. Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

    PubMed

    Jarillo, Javier; Morín, José A; Beltrán-Heredia, Elena; Villaluenga, Juan P G; Ibarra, Borja; Cao, Francisco J

    2017-01-01

    Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.

  9. Ligand Exchange Kinetics of Environmentally Relevant Metals

    SciTech Connect

    Panasci, Adele Frances

    2014-07-15

    The interactions of ground water with minerals and contaminants are of broad interest for geochemists but are not well understood. Experiments on the molecular scale can determine reaction parameters (i.e. rates of ligand exchange, activation entropy, activation entropy, and activation volume) that can be used in computations to gain insight into reactions that occur in natural groundwaters. Experiments to determine the rate of isotopic ligand exchange for three environmentally relevant metals, rhodium (Rh), iron (Fe), and neptunium (Np), are described. Many environmental transformations of metals (e.g. reduction) in soil occur at trivalent centers, Fe(III) in particular. Contaminant ions absorb to mineral surfaces via ligand exchange, and the reversal of this reaction can be dangerous, releasing contaminants into the environment. Ferric iron is difficult to study spectroscopically because most of its complexes are paramagnetic and are generally reactive toward ligand exchange; therefore, Rh(III), which is diamagnetic and less reactive, was used to study substitution reactions that are analogous to those that occur on mineral oxide surfaces. Studies on both Np(V) and Np(VI) are important in their own right, as 237Np is a radioactive transuranic element with a half-life of 2 million years.

  10. Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

    PubMed Central

    Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

    2017-01-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913

  11. (S)-5-(p-Nitrobenzyl)-PCTA, a Promising Bifunctional Ligand with Advantageous Metal Ion Complexation Kinetics

    PubMed Central

    Tircsó, Gyula; Benyó, Enikő Tircsóné; Suh, Eul Hyun; Jurek, Paul; Kiefer, Garry E.; Sherry, A. Dean; Kovács, Zoltán

    2009-01-01

    A bifunctional version of PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) that exhibits fast complexation kinetics with the trivalent lanthanide(III) ions was synthesized in reasonable yields starting from N, N′, N″-tristosyl-(S)-2-(p-nitrobenzyl)-diethylenetriamine. pH-potentiometric studies showed that the basicities of p-nitrobenzyl-PCTA and the parent ligand PCTA were similar. The stability of M(NO2-Bn-PCTA) (M = Mg2+, Ca2+, Cu2+, Zn2+) complexes was similar to that of the corresponding PCTA complexes while the stability of Ln3+ complexes of the bifunctional ligand is somewhat lower than that of PCTA chelates. The rate of complex formation of Ln(NO2-Bn-PCTA) complexes was found to be quite similar to that of PCTA, a ligand known to exhibit the fastest formation rates among all lanthanide macrocyclic ligand complexes studied to date. The acid catalyzed decomplexation kinetic studies of the selected Ln(NO2-Bn-PCTA) complexes showed that the kinetic inertness of the complexes was comparable to that of Ln(DOTA) chelates making the bifunctional ligand NO2-Bn-PCTA suitable for labeling biological vectors with radioisotopes for nuclear medicine applications. PMID:19220012

  12. (S)-5-(p-nitrobenzyl)-PCTA, a promising bifunctional ligand with advantageous metal ion complexation kinetics.

    PubMed

    Tircsó, Gyula; Benyó, Eniko Tircsóné; Suh, Eul Hyun; Jurek, Paul; Kiefer, Garry E; Sherry, A Dean; Kovács, Zoltán

    2009-03-18

    A bifunctional version of PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) that exhibits fast complexation kinetics with the trivalent lanthanide(III) ions was synthesized in reasonable yields starting from N,N',N''-tristosyl-(S)-2-(p-nitrobenzyl)-diethylenetriamine. pH-potentiometric studies showed that the basicities of p-nitrobenzyl-PCTA and the parent ligand PCTA were similar. The stability of M(NO(2)-Bn-PCTA) (M = Mg(2+), Ca(2+), Cu(2+), Zn(2+)) complexes was similar to that of the corresponding PCTA complexes, while the stability of Ln(3+) complexes of the bifunctional ligand is somewhat lower than that of PCTA chelates. The rate of complex formation of Ln(NO(2)-Bn-PCTA) complexes was found to be quite similar to that of PCTA, a ligand known to exhibit the fastest formation rates among all lanthanide macrocyclic ligand complexes studied to date. The acid-catalyzed decomplexation kinetic studies of the selected Ln(NO(2)-Bn-PCTA) complexes showed that the kinetic inertness of the complexes was comparable to that of Ln(DOTA) chelates making the bifunctional ligand NO(2)-Bn-PCTA suitable for labeling biological vectors with radioisotopes for nuclear medicine applications.

  13. Exchange Kinetics of a Hydrophobic Ligand Binding Protein

    NASA Astrophysics Data System (ADS)

    Vaughn, Jeff; Stone, Martin

    2002-03-01

    Conformational fluctuations of proteins are thought to be important for determining the functional roles in biological activity. In some cases, the rates of these conformational changes may be directly correlated to, for example, the rates of catalysis or ligand binding. We are studying the role of conformational fluctuations in the binding of small volatile hydrophobic pheromones by the mouse major urinary proteins (MUPs). Communication among mice occurs, in part, with the MUP-1 protein. This urinary protein binds pheromones as a way to increase the longevity of the pheromone in an extracellular environment. Of interest is that the crystal structure of MUP-1 with a pheromone ligand shows the ligand to be completely occluded from the solvent with no obvious pathway to enter or exit. This suggests that conformational exchange of the protein may be required for ligand binding and release to occur. We hypothesize that the rate of conformational exchange may be a limiting factor determining the rate of ligand association and dissociation. By careful measurement of the on- and off-rates of ligand binding and the rates of conformational changes of the protein, a more defined picture of the interplay between protein structure and function can be obtained. To this end, heteronuclear saturation transfer, ^15N-exchange and ^15N dynamics experiments have been employed to probe the kinetics of ligand binding to MUP-1.

  14. Kinetic isotope effects for fast deuterium and proton exchange rates

    PubMed Central

    Mammoli, Daniele; Kadeřávek, Pavel; Pelupessy, Philippe; Bodenhausen, Geoffrey

    2016-01-01

    By monitoring the effect of deuterium decoupling on the decay of transverse 15N magnetization in D–15N spin pairs during multiple-refocusing echo sequences, we have determined fast D–D exchange rates k D and compared them with fast H–H exchange rates k H in tryptophan to determine the kinetic isotope effect as a function of pH and temperature. PMID:27009684

  15. Kinetic isotope effects for fast deuterium and proton exchange rates.

    PubMed

    Canet, Estel; Mammoli, Daniele; Kadeřávek, Pavel; Pelupessy, Philippe; Bodenhausen, Geoffrey

    2016-04-21

    By monitoring the effect of deuterium decoupling on the decay of transverse (15)N magnetization in D-(15)N spin pairs during multiple-refocusing echo sequences, we have determined fast D-D exchange rates kD and compared them with fast H-H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature.

  16. Active biopolymers confer fast reorganization kinetics.

    PubMed

    Swanson, Douglas; Wingreen, Ned S

    2011-11-18

    Many cytoskeletal biopolymers are "active," consuming energy in large quantities. In this Letter, we identify a fundamental difference between active polymers and passive, equilibrium polymers: for equal mean lengths, active polymers can reorganize faster than equilibrium polymers. We show that equilibrium polymers are intrinsically limited to linear scaling between mean lifetime (or mean first-passage time, or MFPT) and mean length, MFPT∼, by analogy to 1D Potts models. By contrast, we present a simple active-polymer model that improves upon this scaling, such that MFPT∼(1/2). Since, to be biologically useful, structural biopolymers must typically be many monomers long yet respond dynamically to the needs of the cell, the difference in reorganization kinetics may help to justify the active polymers' greater energy cost.

  17. Quantitative Imaging in Laboratory: Fast Kinetics and Fluorescence Quenching

    ERIC Educational Resources Information Center

    Cumberbatch, Tanya; Hanley, Quentin S.

    2007-01-01

    The process of quantitative imaging, which is very commonly used in laboratory, is shown to be very useful for studying the fast kinetics and fluorescence quenching of many experiments. The imaging technique is extremely cheap and hence can be used in many absorption and luminescence experiments.

  18. Quantitative Imaging in Laboratory: Fast Kinetics and Fluorescence Quenching

    ERIC Educational Resources Information Center

    Cumberbatch, Tanya; Hanley, Quentin S.

    2007-01-01

    The process of quantitative imaging, which is very commonly used in laboratory, is shown to be very useful for studying the fast kinetics and fluorescence quenching of many experiments. The imaging technique is extremely cheap and hence can be used in many absorption and luminescence experiments.

  19. Estimation of kinetic and thermodynamic ligand-binding parameters using computational strategies.

    PubMed

    Deganutti, Giuseppe; Moro, Stefano

    2017-03-31

    Kinetic and thermodynamic ligand-protein binding parameters are gaining growing importance as key information to consider in drug discovery. The determination of the molecular structures, using particularly x-ray and NMR techniques, is crucial for understanding how a ligand recognizes its target in the final binding complex. However, for a better understanding of the recognition processes, experimental studies of ligand-protein interactions are needed. Even though several techniques can be used to investigate both thermodynamic and kinetic profiles for a ligand-protein complex, these procedures are very often laborious, time consuming and expensive. In the last 10 years, computational approaches have enormous potential in providing insights into each of the above effects and in parsing their contributions to the changes in both kinetic and thermodynamic binding parameters. The main purpose of this review is to summarize the state of the art of computational strategies for estimating the kinetic and thermodynamic parameters of a ligand-protein binding.

  20. Fast Kinetics of Calcium Signaling and Sensor Design

    PubMed Central

    Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

    2015-01-01

    Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

  1. Kinetic Studies of Reactions in Solution Using Fast Mass Spectrometry

    DTIC Science & Technology

    2013-08-13

    of Reactions in Solution Using Fast Mass Spectrometry Sb. GRANT NUMBER FA9550-10-1-0235 Sc. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Sd . PROJECT...salt and detergent in electrokinetic separations using a spinning disk. We also applied this technique, we believe with great success, to study...chromatography to MS Salts and detergents used in the mobile phase for electro- kinetic separations suppress ionization efficiencies and con

  2. The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands.

    PubMed

    Iloro, Ibon; Narváez, Daniel; Guillén, Nancy; Camacho, Carlos M; Guillén, Lalisse; Cora, Elsa; Pastrana-Ríos, Belinda

    2008-05-15

    Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min(-1) (Tyr), 0.09 min(-1) (Arg, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-sheets). The time constants for AR 0.47 min(-1) (Tyr), 0.04 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (buried 3(10)-helix, beta-turns, and beta-sheets); for HB-EGF 0.89 min(-1) (Tyr), 0.14 min(-1) (Arg and 3(10)-helix), and 1.00 x 10(-3) min(-1) (buried 3(10)-helix, beta-sheets, and beta-turns); and for epiregulin 0.16 min(-1) (Tyr), 0.03 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (3(10)-helix and beta-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.

  3. Lower ligand denticity leading to improved thermodynamic and kinetic stability of the Gd3+ complex: the strange case of OBETA.

    PubMed

    Baranyai, Zsolt; Botta, Mauro; Fekete, Marianna; Giovenzana, Giovanni B; Negri, Roberto; Tei, Lorenzo; Platas-Iglesias, Carlos

    2012-06-18

    OBETA, OBETA, you bet: Thermodynamic and kinetic measurements show an apparent paradox. The stability of complexes of lanthanide trivalent ions is higher with the heptadentate ligand OBETA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) than with its octadentate homologue EGTA (2,2'-oxybis(ethylamine)-N,N,N',N'-tetraacetic acid). The unusual properties of Gd(OBETA)(-) (see structure), combined with the presence of two fast exchanging coordinated water molecules, candidates this complex as an MRI contrast agent. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Kinetics of binding of fluorescent ligands to enzymes with engineered access tunnels.

    PubMed

    Kaushik, Shubhangi; Prokop, Zbynek; Damborsky, Jiri; Chaloupkova, Radka

    2017-01-01

    Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutations introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude, and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method's application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as α/β-hydrolases containing > 100 000 protein sequences based on the Pfam database.

  5. Triglyceride kinetics in fasted and fed E. coli septic rats

    SciTech Connect

    Lanza-Jacoby, S.; Tabares, A. )

    1990-02-26

    The mechanism for the development of hypertriglyceridemia during gram-negative sepsis was studies by examining the liver production and clearance of very-low-density lipoprotein (VLDL) triglyceride (TG). To assess the liver output and peripheral clearance the kinetics of VLDL-TG were determined by a constant intravenous infusion of (2-{sup 3}H) glycerol-labeled VLDL in fasted control, fasted E. coli-treated, fed control, and fed E.coli-treated rats. Lewis inbred rats, 275-300 g, were made septic with 8 {times} 10{sup 7} live E.coli colonies per 100 g body weight. Twenty-four hours following E.coli injection serum TG of fasted E.coli-treated rats was elevated by 170% which was attributed to a 67% decrease in the clearance rate of VLDL-TG in fasted E.coli-treated rats compared with their fasted controls. The secretion of VLDL-TG declined by 31% in the livers of the fasted E.coli-treated rats which was accompanied by a 2-fold increase in the composition of liver TG. In a second series of experiments control and E.coli-treated rats were fed intragastrically (IG) a balanced solution containing glucose plus fat as the sources of nonprotein calories. Serum TG were 26% lower in the fed E.coli-treated rats because the clearance rate increased by 86%. The secretion of TG in the fed septic rats increased by 40% but this difference was not significant. In the septic rat the ability to clear triglycerides from the plasma depends upon the nutritional state.

  6. Organic ligand-induced dissolution kinetics of antimony trioxide.

    PubMed

    Hu, Xingyun; He, Mengchang

    2017-06-01

    The influence of low-molecular-weight dissolved organic matter (LMWDOM) on the dissolution rate of Sb2O3 was investigated. Some representative LMWDOMs with carboxyl, hydroxyl, hydrosulfuryl and amidogen groups occurring naturally in the solution were chosen, namely oxalic acid, citric acid, tartaric acid, EDTA, salicylic acid, phthalandione, glycine, thiolactic acid, xylitol, glucose and catechol. These LMWDOMs were dissolved in inert buffers at pH=3.7, 6.6 and 8.6 and added to powdered Sb2O3 in a stirred, thermostatted reactor (25°C). The addition of EDTA, tartaric acid, thiolactic acid, citric acid and oxalic acid solutions at pH3.7 and catechol at pH8.6 increased the rate of release of antimony. In the 10mmol/L thiolactic acid solution, up to 97% by mass of the antimony was released after 120min reaction. There was no effect on the dissolution of Sb2O3 for the other ligands. A weak correlation between dissolution rate with the dissociation constant of ligands and the stability of the dissolved complex was also found. All the results showed that the extent of the promoting effect of ligands on the dissolution of Sb2O3 was not determined by the stability of the dissolved complex, but by the dissociation constant of ligands and detachment rate of surface chelates from the mineral surface. This study can not only help in further understanding the effect of individual low-molecular-weight organic ligands, but also provides a reference to deduce the effect of natural organic matters with oxygen-bearing functional groups on the dissolution of antimony oxide minerals. Copyright © 2016. Published by Elsevier B.V.

  7. Kinetics of allosteric conformational transition of a macromolecule prior to ligand binding: analysis of stopped-flow kinetic experiments.

    PubMed

    Galletto, Roberto; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2005-01-01

    Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number

  8. A kinetic description of ligand binding to sperm whale myoglobin.

    PubMed

    Gibson, Q H; Olson, J S; McKinnie, R E; Rohlfs, R J

    1986-08-05

    Nanosecond recombination time courses were measured by photolyzing O2, NO, CO, methyl, ethyl, n-propyl, n-butyl, and tert-butyl isocyanide complexes of sperm whale myoglobin with a 30-ns laser pulse at pH 7, 20 degrees C. Absorbance was measured both during and after the excitation pulse and as a function of laser light intensity. The results were analyzed quantitatively in terms of a three-step reaction scheme, MbX in equilibrium B in equilibrium C in equilibrium Mb + X, where Mb is myoglobin, B represents a geminate state in which the ligand is present in the distal pocket but not covalently bound to the iron atom, and C, a state in which the ligand is still embedded in the protein but further away from the heme group. The fitted rate parameters were required to be consistent with the observed overall quantum yield, Q, which had been measured independently using much longer (approximately 0.5 ms) xenon flash pulses. Three major conclusions were derived from these analyses. First, the overall quantum yield of the ligand complex is determined primarily by the competition between the rate of iron-ligand bond formation from the initial photoproduct, kB----MbX, and the rate of migration away from state B, kB----C. For example, kB----C approximately equal to 30-100 microseconds-1 for all three gaseous ligands, whereas both Q and kB----MbX vary over 3 orders of magnitude (i.e. NO, Q = 0.001, kB----MbX approximately equal to 16,000 microseconds-1; O2, Q = 0.1, kB----MbX approximately equal to 500 microseconds-1; CO, Q = 1.0, kB----MbX approximately equal to 2 microseconds-1). Second, for NO, O2, and the isonitriles, the rate-limiting step in the overall association reaction starting from ligand in solution is the formation of state B. The rate constant for this process varies from 2 X 10(7) M-1 s-1 for the gaseous ligands to 0.02-1.4 X 10(5) M-1 s-1 for the isonitriles. In contrast, the B to MbX transition is limiting for CO binding. Third, for all the ligands except CO

  9. Comparison of the kinetics of different Markov models for ligand binding under varying conditions

    SciTech Connect

    Martini, Johannes W. R.; Habeck, Michael

    2015-03-07

    We recently derived a Markov model for macromolecular ligand binding dynamics from few physical assumptions and showed that its stationary distribution is the grand canonical ensemble [J. W. R. Martini, M. Habeck, and M. Schlather, J. Math. Chem. 52, 665 (2014)]. The transition probabilities of the proposed Markov process define a particular Glauber dynamics and have some similarity to the Metropolis-Hastings algorithm. Here, we illustrate that this model is the stochastic analog of (pseudo) rate equations and the corresponding system of differential equations. Moreover, it can be viewed as a limiting case of general stochastic simulations of chemical kinetics. Thus, the model links stochastic and deterministic approaches as well as kinetics and equilibrium described by the grand canonical ensemble. We demonstrate that the family of transition matrices of our model, parameterized by temperature and ligand activity, generates ligand binding kinetics that respond to changes in these parameters in a qualitatively similar way as experimentally observed kinetics. In contrast, neither the Metropolis-Hastings algorithm nor the Glauber heat bath reflects changes in the external conditions correctly. Both converge rapidly to the stationary distribution, which is advantageous when the major interest is in the equilibrium state, but fail to describe the kinetics of ligand binding realistically. To simulate cellular processes that involve the reversible stochastic binding of multiple factors, our pseudo rate equation model should therefore be preferred to the Metropolis-Hastings algorithm and the Glauber heat bath, if the stationary distribution is not of only interest.

  10. Kinetics of Fast Atoms in the Terrestrial Atmosphere

    NASA Technical Reports Server (NTRS)

    Kharchenko, Vasili A.; Dalgarno, A.; Mellott, Mary (Technical Monitor)

    2002-01-01

    This report summarizes our investigations performed under NASA Grant NAG5-8058. The three-year research supported by the Geospace Sciences SR&T program (Ionospheric, Thermospheric, and Mesospheric Physics) has been designed to investigate fluxes of energetic oxygen and nitrogen atoms in the terrestrial thermosphere. Fast atoms are produced due to absorption of the solar radiation and due to coupling between the ionosphere and the neutral thermospheric gas. We have investigated the impact of hot oxygen and nitrogen atoms on the thermal balance, chemistry and radiation properties of the terrestrial thermosphere. Our calculations have been focused on the accurate quantitative description of the thermalization of O and N energetic atoms in collisions with atom and molecules of the ambient neutral gas. Upward fluxes of oxygen and nitrogen atoms, the rate of atmospheric heating by hot oxygen atoms, and the energy input into translational and rotational-vibrational degrees of atmospheric molecules have been evaluated. Altitude profiles of hot oxygen and nitrogen atoms have been analyzed and compared with available observational data. Energetic oxygen atoms in the terrestrial atmosphere have been investigated for decades, but insufficient information on the kinetics of fast atmospheric atoms has been a main obstacle for the interpretation of observational data and modeling of the hot geocorona. The recent development of accurate computational methods of the collisional kinetics is seen as an important step in the quantitative description of hot atoms in the thermosphere. Modeling of relaxation processes in the terrestrial atmosphere has incorporated data of recent observations, and theoretical predictions have been tested by new laboratory measurements.

  11. Crystal surface mediated structure transformation of a kinetic framework composed of multi-interactive ligand TPHAP and Co(II).

    PubMed

    Yakiyama, Yumi; Ueda, Akira; Morita, Yasushi; Kawano, Masaki

    2012-11-07

    A tripyridyl multi-interactive ligand TPHAP is prepared by a one-pot reaction on a gram scale. Network formation of Co(ii) with TPHAP(-) gave kinetic and thermally more stable products. The kinetic network showed an unprecedented dynamic network transformation on the crystal surface by a ligand exchange reaction.

  12. Microdroplet fusion mass spectrometry for fast reaction kinetics

    PubMed Central

    Lee, Jae Kyoo; Kim, Samuel; Nam, Hong Gil; Zare, Richard N.

    2015-01-01

    We investigated the fusion of high-speed liquid droplets as a way to record the kinetics of liquid-phase chemical reactions on the order of microseconds. Two streams of micrometer-size droplets collide with one another. The droplets that fused (13 μm in diameter) at the intersection of the two streams entered the heated capillary inlet of a mass spectrometer. The mass spectrum was recorded as a function of the distance x between the mass spectrometer inlet and the droplet fusion center. Fused droplet trajectories were imaged with a high-speed camera, revealing that the droplet fusion occurred approximately within a 500-μm radius from the droplet fusion center and both the size and the speed of the fused droplets remained relatively constant as they traveled from the droplet fusion center to the mass spectrometer inlet. Evidence is presented that the reaction effectively stops upon entering the heated inlet of the mass spectrometer. Thus, the reaction time was proportional to x and could be measured and manipulated by controlling the distance x. Kinetic studies were carried out in fused water droplets for acid-induced unfolding of cytochrome c and hydrogen–deuterium exchange in bradykinin. The kinetics of the former revealed the slowing of the unfolding rates at the early stage of the reaction within 50 μs. The hydrogen–deuterium exchange revealed the existence of two distinct populations with fast and slow exchange rates. These studies demonstrated the power of this technique to detect reaction intermediates in fused liquid droplets with microsecond temporal resolution. PMID:25775573

  13. Microdroplet fusion mass spectrometry for fast reaction kinetics.

    PubMed

    Lee, Jae Kyoo; Kim, Samuel; Nam, Hong Gil; Zare, Richard N

    2015-03-31

    We investigated the fusion of high-speed liquid droplets as a way to record the kinetics of liquid-phase chemical reactions on the order of microseconds. Two streams of micrometer-size droplets collide with one another. The droplets that fused (13 μm in diameter) at the intersection of the two streams entered the heated capillary inlet of a mass spectrometer. The mass spectrum was recorded as a function of the distance x between the mass spectrometer inlet and the droplet fusion center. Fused droplet trajectories were imaged with a high-speed camera, revealing that the droplet fusion occurred approximately within a 500-μm radius from the droplet fusion center and both the size and the speed of the fused droplets remained relatively constant as they traveled from the droplet fusion center to the mass spectrometer inlet. Evidence is presented that the reaction effectively stops upon entering the heated inlet of the mass spectrometer. Thus, the reaction time was proportional to x and could be measured and manipulated by controlling the distance x. Kinetic studies were carried out in fused water droplets for acid-induced unfolding of cytochrome c and hydrogen-deuterium exchange in bradykinin. The kinetics of the former revealed the slowing of the unfolding rates at the early stage of the reaction within 50 μs. The hydrogen-deuterium exchange revealed the existence of two distinct populations with fast and slow exchange rates. These studies demonstrated the power of this technique to detect reaction intermediates in fused liquid droplets with microsecond temporal resolution.

  14. Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

    PubMed Central

    El Hassan, G. A.; Zablotowicz, R. M.; Focht, D. D.

    1985-01-01

    Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO3−, NO2−, and N2O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (Ks) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: Ks, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq−1; μmax, 0.41 and 0.33 h−1. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent Ks values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the Ks for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O2 is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μmax, 0.41 h−1) when A26 was grown with either O2 or NO3− as an electron acceptor and were only slightly reduced when A28 was grown with NO3− (0.25 h−1) as opposed to O2 (0.33 h−1). PMID:16346745

  15. Copper(II) complexes of quinoline polyazamacrocyclic scorpiand-type ligands: X-ray, equilibrium and kinetic studies.

    PubMed

    Castillo, Carmen E; Angeles Máñez, M; Basallote, Manuel G; Paz Clares, M; Blasco, Salvador; García-España, Enrique

    2012-05-14

    The formation of Cu(II) complexes with two isomeric quinoline-containing scorpiand-type ligands has been studied. The ligands have a tetraazapyridinophane core appended with an ethylamino tail including 2-quinoline (L1) or 4-quinoline (L2) functionalities. Potentiometric studies indicate the formation of stable CuL(2+) species with both ligands, the L1 complex being 3-4 log units more stable than the L2 complex. The crystal structure of [Cu(L1)](ClO(4))(2)·H(2)O shows that the coordination geometry around the Cu(2+) ions is distorted octahedral with significant axial elongation; the four Cu-N distances in the equatorial plane vary from 1.976 to 2.183 Å, while the axial distances are of 2.276 and 2.309 Å. The lower stability of the CuL2(2+) complex and its capability of forming protonated and hydroxo complexes suggest a penta-dentate coordination of the ligand, in agreement with the type of substitution at the quinoline ring. Kinetic studies on complex formation can be interpreted by considering that initial coordination of L1 and L2 takes place through the nitrogen atom in the quinoline ring. This is followed by coordination of the remaining nitrogen atoms, in a process that is faster in the L1 complex probably because substitution at the quinoline ring facilitates the reorganization. Kinetic studies on complex decomposition provide clear evidence on the occurrence of the molecular motion typical of scorpiands in the case of the L2 complex, for which decomposition starts with a very fast process (sub-millisecond timescale) that involves a shift in the absorption band from 643 to 690 nm.

  16. Kinetics of membrane adhesion mediated by ligand-receptor interaction studied with a biomimetic system.

    PubMed Central

    Boulbitch, A; Guttenberg, Z; Sackmann, E

    2001-01-01

    We report the first measurement of the kinetics of adhesion of a single giant vesicle controlled by the competition between membrane-substrate interaction mediated by ligand-receptor interaction, gravitation, and Helfrich repulsion. To model the cell-tissue interaction, we doped the vesicles with lipid-coupled polymers (mimicking the glycocalix) and the reconstituted ligands selectively recognized by alpha(IIb)beta(3) integrin-mediating specific attraction forces. The integrin was grafted on glass substrates to act as a target cell. The adhesion of the vesicle membrane to the integrin-covered surface starts with the spontaneous formation of a small (approximately 200 nm) domain of tight adhesion, which then gradually grows until the whole adhesion area is in the state of tight adhesion. The time of adhesion varies from few tens of seconds to about one hour depending on the ligand and lipopolymer concentration. At small ligand concentrations, we observed the displacement xi of the front of tight adhesion following the square root law xi approximately t(1/2), whereas, at high concentrations, we found a linear law xi approximately t. We show both experimentally and theoretically that the t(1/2)-regime is dominated by diffusion of ligands, and the xi approximately t-regime by the kinetics of ligands-receptors association. PMID:11606287

  17. Ligand-receptor binding kinetics in surface plasmon resonance cells: a Monte Carlo analysis

    NASA Astrophysics Data System (ADS)

    Carroll, Jacob; Raum, Matthew; Forsten-Williams, Kimberly; Täuber, Uwe C.

    2016-12-01

    Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of spatio-temporal correlations in binary reaction kinetics in SPR cell geometries, and demonstrate the failure of a mean-field analysis of SPR cells in the regime of high Damköhler number {{Da}}\\gt 0.1, where the spatio-temporal correlations due to diffusive transport and ligand-receptor rebinding events dominate the dynamics of SPR systems.

  18. Laminar-flow fluid mixer for fast fluorescence kinetics studies.

    PubMed Central

    Pabit, Suzette A; Hagen, Stephen J

    2002-01-01

    The ability to mix aqueous liquids on microsecond time scales, while consuming minimal amounts of sample and maintaining UV-visible optical access to the mixing region, is highly desirable for a range of biophysical studies of fast protein and nucleic acid interactions and folding. We have constructed a laminar coaxial jet mixer that allows the measurement of UV-excited fluorescence from nanoliter and microliter quantities of material, mixed at microsecond rates. The mixer injects a narrow cylindrical stream (radius a < 1 microm) of fluorescent sample into a larger flow of diluting buffer that moves through a capillary (100 microm i.d.) at a speed approximately 20 cm/s, under laminar flow conditions (Re approximately equal to 14). Construction from a fused silica capillary allows the laser excitation (at 266 nm) and detection (at 350 nm) of tryptophan fluorescence at reasonably low working concentrations, without interference from background fluorescence. Using this mixer we have measured sub-millisecond fluorescence quenching kinetics while consuming fluorescent sample at rates no greater than 6 nl/s. Consumption of the diluting buffer is also very modest (approximately 1-3 microl/s) in comparison with other rapid mixer designs. PMID:12414719

  19. Gompertz kinetics model of fast chemical neurotransmission currents.

    PubMed

    Easton, Dexter M

    2005-10-01

    At a chemical synapse, transmitter molecules ejected from presynaptic terminal(s) bind reversibly with postsynaptic receptors and trigger an increase in channel conductance to specific ions. This paper describes a simple but accurate predictive model for the time course of the synaptic conductance transient, based on Gompertz kinetics. In the model, two simple exponential decay terms set the rates of development and decline of transmitter action. The first, r, triggering conductance activation, is surrogate for the decelerated rate of growth of conductance, G. The second, r', responsible for Y, deactivation of the conductance, is surrogate for the decelerated rate of decline of transmitter action. Therefore, the differential equation for the net conductance change, g, triggered by the transmitter is dg/dt=g(r-r'). The solution of that equation yields the product of G(t), representing activation, and Y(t), which defines the proportional decline (deactivation) of the current. The model fits, over their full-time course, published records of macroscopic ionic current associated with fast chemical transmission. The Gompertz model is a convenient and accurate method for routine analysis and comparison of records of synaptic current and putative transmitter time course. A Gompertz fit requiring only three independent rate constants plus initial current appears indistinguishable from a Markov fit using seven rate constants.

  20. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel.

    PubMed

    Laha, Kurt T; Ghosh, Borna; Czajkowski, Cynthia

    2013-01-01

    Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9 ± 5.1 ms and 1.2 ± 0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3 ± 0.3 s and deactivated even slower with a time constant of 4.6 ± 1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying p

  1. Photoperturbation-relaxation approach to the kinetics of cooperative ligand binding by heme proteins

    SciTech Connect

    Schuresko, D.D.

    1983-05-01

    A small perturbation technique for measuring the ligand photodissociation and recombination kinetics of heme proteins has been developed. The Photodissociation Perturbation Relaxation (PPR) method involves perturbing the photodissociation rates of ligand-heme systems maintained at photointensity-dependent, nonequilibrium photostationary states. The theoretical and experimental datails of the PPR method are presented herein. A formalism for computing PPR amplitudes and time constants for complex reaction mechanisms for the eigenvectors and eigenvalues of the appropriate linear rate equations is derived; a FORTRAN code embodying this formalism is presented. PPR kinetics measurements obtained for the carbon monoxide derivatives of sperm whale myoglobin and human hemoglobin are presented. CO-hemoglobin combination rate constants and photodissociation quantum efficiencies, determined via fitting model-derived relaxation eigenmodes to PPR transients are presented.

  2. Protein conformational plasticity and complex ligand-binding kinetics explored by atomistic simulations and Markov models

    PubMed Central

    Plattner, Nuria; Noé, Frank

    2015-01-01

    Understanding the structural mechanisms of protein–ligand binding and their dependence on protein sequence and conformation is of fundamental importance for biomedical research. Here we investigate the interplay of conformational change and ligand-binding kinetics for the serine protease Trypsin and its competitive inhibitor Benzamidine with an extensive set of 150 μs molecular dynamics simulation data, analysed using a Markov state model. Seven metastable conformations with different binding pocket structures are found that interconvert at timescales of tens of microseconds. These conformations differ in their substrate-binding affinities and binding/dissociation rates. For each metastable state, corresponding solved structures of Trypsin mutants or similar serine proteases are contained in the protein data bank. Thus, our wild-type simulations explore a space of conformations that can be individually stabilized by adding ligands or making suitable changes in protein sequence. These findings provide direct evidence of conformational plasticity in receptors. PMID:26134632

  3. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory.

    PubMed

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

    2015-12-28

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  4. Role of Desolvation in Thermodynamics and Kinetics of Ligand Binding to a Kinase

    PubMed Central

    2015-01-01

    Computer simulations are used to determine the free energy landscape for the binding of the anticancer drug Dasatinib to its src kinase receptor and show that before settling into a free energy basin the ligand must surmount a free energy barrier. An analysis based on using both the ligand-pocket separation and the pocket-water occupancy as reaction coordinates shows that the free energy barrier is a result of the free energy cost for almost complete desolvation of the binding pocket. The simulations further show that the barrier is not a result of the reorganization free energy of the binding pocket. Although a continuum solvent model gives the location of free energy minima, it is not able to reproduce the intermediate free energy barrier. Finally, it is shown that a kinetic model for the on rate constant in which the ligand diffuses up to a doorway state and then surmounts the desolvation free energy barrier is consistent with published microsecond time-scale simulations of the ligand binding kinetics for this system [Shaw, D. E. et al. J. Am. Chem. Soc.2011, 133, 9181−918321545110]. PMID:25516727

  5. Impact of human galectin-1 binding to saccharide ligands on dimer dissociation kinetics and structure.

    PubMed

    Romero, Juan M; Trujillo, Madia; Estrin, Darío A; Rabinovich, Gabriel A; Di Lella, Santiago

    2016-12-01

    Endogenous lectins can control critical biological responses, including cell communication, signaling, angiogenesis and immunity by decoding glycan-containing information on a variety of cellular receptors and the extracellular matrix. Galectin-1 (Gal-1), a prototype member of the galectin family, displays only one carbohydrate recognition domain and occurs in a subtle homodimerization equilibrium at physiologic concentrations. Such equilibrium critically governs the function of this lectin signaling by allowing tunable interactions with a preferential set of glycosylated receptors. Here, we used a combination of experimental and computational approaches to analyze the kinetics and mechanisms connecting Gal-1 ligand unbinding and dimer dissociation processes. Kinetic constants of both processes were found to differ by an order of magnitude. By means of steered molecular dynamics simulations, the ligand unbinding process was followed monitoring water occupancy changes. By determining the water sites in a carbohydrate binding place during the unbinding process, we found that rupture of ligand-protein interactions induces an increase in energy barrier while ligand unbinding process takes place, whereas the entry of water molecules to the binding groove and further occupation of their corresponding water sites contributes to lowering of the energy barrier. Moreover, our findings suggested local asymmetries between the two subunits in the dimer structure detected at a nanosecond timescale. Thus, integration of experimental and computational data allowed a more complete understanding of lectin ligand binding and dimerization processes, suggesting new insights into the relationship between Gal-1 structure and function and renewing the discussion on the biophysics and biochemistry of lectin-ligand lattices.

  6. Theory of Kinetics of Multistep Ligand-Receptor Assembly in Dissipating and Fluctuating Environments

    NASA Astrophysics Data System (ADS)

    Teslenko, Victor I.; Kapitanchuk, Oleksiy L.

    2013-09-01

    Multistep kinetic processes play a key role in physics (excitation transfer, energy degradation), chemistry (ligand-receptor assembly, radical reactions) and biology (signal perception, molecular recognition). While a phenomenological thermodynamic approach for modeling the elementary acts of transitions underlying the maintaining of a system's stationary and equilibrium states is now well recognized, a more satisfying microscopic description based on the consistent understanding of dissipation and fluctuation processes accompanying the multistep relaxations remains elusive. In this paper, a microscopic theory of kinetics of a few-state system exhibited the energy fluctuations and coupled to a condensed medium is developed. The theory is formulated such as of being an example of the case of irreversible multistep ligand-receptor assembly in a dissipating environment. We first derive general expression for the probability of transitions between the system states valid on the whole timescale and then reduce this expression to the effectively slow times by making it an average over both the steady-state fluctuations of a system's energies and the equilibrium vibrations of the environment. Further, we calculate the populations of states for the sequence of cases of the three-to-two-to-single-step assemblage in dependence on the temperature, viscosity and ligand concentration. Finally, we discuss the results obtained with reference to the case of "negative" cooperativity emerging by virtue of the irreversibility of the last kinetic step.

  7. Ligand recombination and a hierarchy of solvent slaved dynamics: the origin of kinetic phases in hemeproteins.

    PubMed

    Samuni, Uri; Dantsker, David; Roche, Camille J; Friedman, Joel M

    2007-08-15

    Ligand recombination studies play a central role both for characterizing different hemeproteins and their conformational states but also for probing fundamental biophysical processes. Consequently, there is great importance to providing a foundation from which one can understand the physical processes that give rise to and modulate the large range of kinetic patterns associated with ligand recombination in myoglobins and hemoglobins. In this work, an overview of cryogenic and solution phase recombination phenomena for COMb is first reviewed and then a new paradigm is presented for analyzing the temperature and viscosity dependent features of kinetic traces in terms of multiple phases that reflect which tier(s) of solvent slaved protein dynamics is (are) operative on the photoproduct population during the time course of the measurement. This approach allows for facile inclusion of both ligand diffusion among accessible cavities and conformational relaxation effects. The concepts are illustrated using kinetic traces and MEM populations derived from the CO recombination process for wild type and mutant myoglobins either in sol-gel matrices bathed in glycerol or in trehalose-derived glassy matrices.

  8. Oriented attachment kinetics for ligand capped nanocrystals: coarsening of thiol-PbS nanoparticles.

    PubMed

    Zhang, Jing; Wang, Yonghao; Zheng, Jinsheng; Huang, Feng; Chen, Dagui; Lan, Youzhao; Ren, Guoqiang; Lin, Zhang; Wang, Chen

    2007-02-15

    In this work, the growth kinetics of thiol-capped PbS nanoparticles was studied. Two-stage growth process was observed, which was controlled first by oriented attachment (OA) mechanism and then by the hybrid Ostwald ripening (OR) and OA mechanism. Different from the NaOH-ZnS system, where OA will occur between any two multilevel nanoparticles, an OA kinetic model only considering the attachment related to original particles was fitted well with the experimental results. Analysis reveals that this model may be a universal one to describe the OA crystal growth process of nanocrystals capped with easily destroyed ligands, such as thiol-ZnS in the previous report. The OA crystal growth characteristics determined by the surface agent were discussed and compared. We propose that with stronger surface capping, the OR growth of nanocrystals is hindered, which facilitates the size controlling via OA kinetics during nanosynthesis.

  9. Resolving the Fast Kinetics of Cooperative Binding: Ca2+ Buffering by Calretinin

    PubMed Central

    Faas, Guido C; Schwaller, Beat; Vergara, Julio L; Mody, Istvan

    2007-01-01

    Cooperativity is one of the most important properties of molecular interactions in biological systems. It is the ability to influence ligand binding at one site of a macromolecule by previous ligand binding at another site of the same molecule. As a consequence, the affinity of the macromolecule for the ligand is either decreased (negative cooperativity) or increased (positive cooperativity). Over the last 100 years, O2 binding to hemoglobin has served as the paradigm for cooperative ligand binding and allosteric modulation, and four practical models were developed to quantitatively describe the mechanism: the Hill, the Adair-Klotz, the Monod-Wyman-Changeux, and the Koshland-Némethy-Filmer models. The predictions of these models apply under static conditions when the binding reactions are at equilibrium. However, in a physiological setting, e.g., inside a cell, the timing and dynamics of the binding events are essential. Hence, it is necessary to determine the dynamic properties of cooperative binding to fully understand the physiological implications of cooperativity. To date, the Monod-Wyman-Changeux model was applied to determine the kinetics of cooperative binding to biologically active molecules. In this model, cooperativity is established by postulating two allosteric isoforms with different binding properties. However, these studies were limited to special cases, where transition rates between allosteric isoforms are much slower than the binding rates or where binding and unbinding rates could be measured independently. For all other cases, the complex mathematical description precludes straightforward interpretations. Here, we report on calculating for the first time the fast dynamics of a cooperative binding process, the binding of Ca2+ to calretinin. Calretinin is a Ca2+-binding protein with four cooperative binding sites and one independent binding site. The Ca2+ binding to calretinin was assessed by measuring the decay of free Ca2+ using a fast

  10. Fast O2 Binding at Dicopper Complexes Containing Schiff-Base Dinucleating Ligands

    PubMed Central

    Company, Anna; Gómez, Laura; Mas-Ballesté, Rubén; Korendovych, Ivan V.; Ribas, Xavi; Poater, Albert; Parella, Teodor; Fontrodona, Xavier; Benet-Buchholz, Jordi; Solà, Miquel; Que, Lawrence; Rybak-Akimova, Elena; Costas, Miquel

    2008-01-01

    A new family of dicopper(I) complexes [CuI2RL](X)2, (R = H, 1X, R = tBu, 2X and R = NO2, 3X, X = CF3SO3, ClO4, SbF6 or BArF, BArF = [B{3,5-(CF3)2-C6H3}4]−), where RL is a Schiff-base ligand containing two tridentate binding sites linked by a xylyl spacer have been prepared, characterized, and their reaction with O2 studied. The complexes were designed with the aim of reproducing structural aspects of the active site of type 3 dicopper proteins; they contain two three-coordinate copper sites and a rather flexible podand ligand backbone. The solid state structures of 1ClO4, 2CF3SO3, 2ClO4 and 3BArF·CH3CN have been established by single crystal X-ray diffraction analysis. 1ClO4 adopts a polymeric structure in solution while 2CF3SO3, 2ClO4 and 3BArF·CH3CN are monomeric. The complexes have been studied in solution by means of 1H and 19F NMR spectroscopy, which put forward the presence of dynamic processes in solution. 1-3BArF and 1-3CF3SO3 in acetone react rapidly with O2 to generate metaestable [CuIII2(μ-O)2(RL)]2+ 1-3(O2) and [CuIII2(μ-O)2(CF3SO3)(RL)]+ 1-3(O2)(CF3SO3) species, respectively that have been characterized by UV-vis spectroscopy and resonance Raman analysis. Instead, reaction of 1-3BArF with O2 in CH2Cl2 results in intermolecular O2 binding. DFT methods have been used to study the chemical identities and structural parameters of the O2 adducts, and the relative stability of the CuIII2(μ-O)2 form with respect to the CuII2(μ-η2: η2-peroxo) isomer. The reaction of 1X, X = CF3SO3 and BArF with O2 in acetone has been studied by stopped-flow exhibiting an unexpected very fast reaction rate (k = 3.82(4) × 103 M−1s−1, ΔH‡ = 4.9 ± 0.5 kJ·mol−1, ΔS‡ = −148 ± 5 J·K−1·mol−1), nearly three orders of magnitude faster than in the parent [CuI2(m-XYLMeAN)]2+. Thermal decomposition of 1-3(O2) does not result in aromatic hydroxylation. The mechanism and kinetics of O2 binding to 1X (X = CF3SO3 and BArF) is discussed and compared with those

  11. Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

    PubMed Central

    Chesla, S E; Selvaraj, P; Zhu, C

    1998-01-01

    We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2. PMID:9726957

  12. Slow VO2 off-kinetics in skeletal muscle is associated with fast PCr off-kinetics--and inversely.

    PubMed

    Korzeniewski, Bernard; Zoladz, Jerzy A

    2013-09-01

    The computer model of the bioenergetic system in skeletal muscle, developed previously, was used to study the effect of the characteristic decay time of the parallel activation of oxidative phosphorylation [τ(OFF)] during muscle recovery on the muscle oxygen consumption rate (Vo2) and phosphocreatine (PCr) work-to-rest transition (off)-kinetics and on the relationship between the Vo2 and PCr rest-to-work transition (on)- and off-kinetics in moderate and heavy exercise. An increase in τ(OFF) slows down the initial phase of the muscle Vo2 off-kinetics and accelerates the PCr off-kinetics. As a result, the relationship between the initial phase of the Vo2 off-kinetics (lasting approximately 3-60 s in computer simulations) and the PCr off-kinetics is inverse: the slower the former, the faster the latter. A faster initial phase of the Vo2 off-kinetics is associated with a slower late phase of the Vo2 off-kinetics, and as a result, the integral of Vo2 above baseline during recovery, representing the oxygen debt, is identical in all cases [values of τ(OFF)] for a given PCr decrease. Depending on τ(OFF), the muscle Vo2 on-kinetics was either equally fast or slower than the Vo2 off-kinetics in moderate exercise and always slower in heavy exercise. PCr on-kinetics was always faster than PCr off-kinetics. This study clearly demonstrates that τ(OFF) has a pronounced impact on the mutual relations between the muscle Vo2 and PCr on- and off-kinetics.

  13. Simple and fast screening of G-quadruplex ligands with electrochemical detection system.

    PubMed

    Fan, Qiongxuan; Li, Chao; Tao, Yaqin; Mao, Xiaoxia; Li, Genxi

    2016-11-01

    Small molecules that may facilitate and stabilize the formation of G-quadruplexes can be used for cancer treatments, because the G-quadruplex structure can inhibit the activity of telomerase, an enzyme over-expressed in many cancer cells. Therefore, there is considerable interest in developing a simple and high-performance method for screening small molecules binding to G-quadruplex. Here, we have designed a simple electrochemical approach to screen such ligands based on the fact that the formation and stabilization of G-quadruplex by ligand may inhibit electron transfer of redox species to electrode surface. As a proof-of-concept study, two types of classical G-quadruplex ligands, TMPyP4 and BRACO-19, are studied in this work, which demonstrates that this method is fast and robust and it may be applied to screen G-quadruplex ligands for anticancer drugs testing and design in the future. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    PubMed Central

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD <10−8 M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  15. Fast pulling of ligand approach for the design of β-secretase 1 inhibitors

    NASA Astrophysics Data System (ADS)

    Truong, Duc Toan; Nguyen, Minh Tung; Vu, Van V.; Ngo, Son Tung

    2017-03-01

    The fast pulling of ligand (FPL) method, which evaluates the relative ligand-protein binding affinity with low CPU usage and high accuracy, was applied for the first time to determine the affinity of β-secretase 1 (BACE1) and its inhibitors using steered-molecular dynamics simulations. The total non-bonded interaction energy difference ΔEtotal is a highly appropriate criterion to predict the relative BACE1-inhibitor binding affinity with strong correlation to experimental data (R = 0.92) and small deviation (δEtotal = 7 %). The van der Waals interaction and electrostatic interaction contribute 56% and 44% to the total non-bonded interaction energy between BACE1 and its inhibitors.

  16. Pb(2+) complexes of small-cavity azamacrocyclic ligands: thermodynamic and kinetic studies.

    PubMed

    Liberato, A; Aguinaco, A; Clares, M P; Delgado-Pinar, E; Pitarch-Jarque, J; Blasco, S; Basallote, M G; García-España, E; Verdejo, B

    2017-05-23

    The synthesis, acid-base behavior and Pb(2+) coordination chemistry of the new aza-scorpiand like ligand 5-[2-(N-2-fluorenyl)ethylamino]-2,5,8-triaza[9]-2,6-pyridinophane (L1) have been studied by potentiometry, NMR and spectrofluorimetric titrations, and the results are compared with those obtained for the related compounds L2, lacking the fluorenyl group, and L3, the macrocycle lacking the pendant arm. The crystal structures obtained for complexes [PbL1][PbL1Cl](NO3)Cl2·4H2O (1) and [PbL3](ClO4)2 (2) reveal that the metal ion is located over the plane defined by the nitrogen atoms of the macrocyclic core due to its inability to accommodate the large Pb(2+) ion in the macrocyclic cavity. For L1, the secondary amino group of the pendant arm is implicated in the coordination of the metal ion, although the stereoactive lone pair of Pb(2+) prevents the closed conformation associated with the coordination of metal ions in aza-scorpiand derivatives. The kinetics of the acid-promoted dissociation of the ligand from the Pb(2+) complexes with the three ligands have been studied using stopped-flow with simultaneous absorbance and fluorescence detection. The results indicate that in spite of their similarity, the dissociation of the metal ion occurs with very different rates in the three complexes. During the course of the kinetic studies evidence was obtained for the occurrence of a photochemical process that leads to ligand degradation with the unexpected elimination of one CH2CH2 fragment from the macrocyclic core.

  17. Colloidal nanoparticle size control: experimental and kinetic modeling investigation of the ligand-metal binding role in controlling the nucleation and growth kinetics.

    PubMed

    Mozaffari, Saeed; Li, Wenhui; Thompson, Coogan; Ivanov, Sergei; Seifert, Soenke; Lee, Byeongdu; Kovarik, Libor; Karim, Ayman M

    2017-09-21

    Despite the major advancements in colloidal metal nanoparticles synthesis, a quantitative mechanistic treatment of the ligand's role in controlling their size remains elusive. We report a methodology that combines in situ small angle X-ray scattering (SAXS) and kinetic modeling to quantitatively capture the role of ligand-metal binding (with the metal precursor and the nanoparticle surface) in controlling the synthesis kinetics. We demonstrate that accurate extraction of the kinetic rate constants requires using both, the size and number of particles obtained from in situ SAXS to decouple the contributions of particle nucleation and growth to the total metal reduction. Using Pd acetate and trioctylphosphine in different solvents, our results reveal that the binding of ligands with both the metal precursor and nanoparticle surface play a key role in controlling the rates of nucleation and growth and consequently the final size. We show that the solvent can affect the metal-ligand binding and consequently ligand coverage on the nanoparticles surface which has a strong effect on the growth rate and final size (1.4 nm in toluene and 4.3 nm in pyridine). The proposed kinetic model quantitatively predicts the effects of varying the metal concentration and ligand/metal ratio on nanoparticle size for our work and literature reports. More importantly, we demonstrate that the final size is exclusively determined by the nucleation and growth kinetics at early times and not how they change with time. Specifically, the nanoparticle size in this work and many literature reports can be predicted using a single, model independent kinetic descriptor, (growth-to-nucleation rate ratio)(1/3), despite the different metals and synthetic conditions. The proposed model and kinetic descriptor could serve as powerful tools for the design of colloidal nanoparticles with specific sizes.

  18. Scaling of Kinetic Instability Induced Fast Ion Losses in NSTX

    SciTech Connect

    E.D. Fredrickson; D. Darrow; S. Medley; J. Menard; H. Park; L. Roquemore; D. Stutman; K. Tritz; S. Kubota; K.C. Lee

    2005-06-24

    During neutral beam injection (NBI) in the National Spherical Torus Experiment (NSTX), a wide variety of fast ion driven instabilities is excited by the large ratio of fast ion velocity to Alfven velocity, together with the relatively high fast ion beta, beta(sub)f. The fast ion instabilities have frequencies ranging from a few kilohertz to the ion cyclotron frequency. The modes can be divided roughly into three categories, starting with Energetic Particle Modes (EPM) in the lowest frequency range (0 to 120 kHz), the Toroidal Alfven Eigenmodes (TAE) in the intermediate frequency range (50 to 200 kHz) and the Compressional and Global Alfven Eigenmodes (CAE and GAE, respectively) from approximately equal to 300 kHz up to the ion cyclotron frequency. Each of these categories of modes exhibits a wide range of behavior, including quasi-continuous oscillation, bursting, chirping and, except for the lower frequency range, turbulence.

  19. Fast algorithms for combustion kinetics calculations: A comparison

    NASA Technical Reports Server (NTRS)

    Radhakrishnan, K.

    1984-01-01

    To identify the fastest algorithm currently available for the numerical integration of chemical kinetic rate equations, several algorithms were examined. Findings to date are summarized. The algorithms examined include two general-purpose codes EPISODE and LSODE and three special-purpose (for chemical kinetic calculations) codes CHEMEQ, CRK1D, and GCKP84. In addition, an explicit Runge-Kutta-Merson differential equation solver (IMSL Routine DASCRU) is used to illustrate the problems associated with integrating chemical kinetic rate equations by a classical method. Algorithms were applied to two test problems drawn from combustion kinetics. These problems included all three combustion regimes: induction, heat release and equilibration. Variations of the temperature and species mole fraction are given with time for test problems 1 and 2, respectively. Both test problems were integrated over a time interval of 1 ms in order to obtain near-equilibration of all species and temperature. Of the codes examined in this study, only CREK1D and GCDP84 were written explicitly for integrating exothermic, non-isothermal combustion rate equations. These therefore have built-in procedures for calculating the temperature.

  20. Colloidal nanoparticle size control: Experimental and kinetic modeling investigation of the ligand-metal binding role in controlling the nucleation and growth kinetics

    DOE PAGES

    Mozaffari, Saeed; Li, Wenhui; Thompson, Coogan; ...

    2017-09-01

    Despite the major advancements in colloidal metal nanoparticles synthesis, a quantitative mechanistic treatment of the ligand’s role in controlling their size remains elusive. We report a methodology that combines in-situ small angle x-ray scattering (SAXS) and kinetic modeling to quantitatively capture the role of ligand-metal binding (with the metal precursor and the nanoparticle surface) in controlling the synthesis kinetics. We demonstrate that accurate extraction of the kinetic rate constants requires using both, the size and number of particles obtained from in-situ SAXS to decouple the contributions of particle nucleation and growth to the total metal reduction. Using Pd acetate andmore » trioctylphosphine in different solvents, our results reveal that the binding of ligands with both the metal precursor and nanoparticle surface play a key role in controlling the rates of nucleation and growth and consequently the final size. We show that the solvent can affect the metal-ligand binding and consequently ligand coverage on the nanoparticles surface which has a strong effect on the growth rate and final size (1.4 nm in toluene and 4.3 nm in pyridine). The proposed kinetic model quantitatively predicts the effects of varying the metal concentration and ligand/metal ratio on nanoparticle size for our work and literature reports. More importantly, we demonstrate that the final size is exclusively determined by the nucleation and growth kinetics at early times and not how they change with time. Specifically, the nanoparticle size in this work and many literature reports can be predicted using a single, model independent kinetic descriptor, (Growth-to-Nucleation rate ratio)1/3, despite the different metals and synthetic conditions. The proposed model and kinetic descriptor could serve as powerful tools for the design of colloidal nanoparticles with specific sizes.« less

  1. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain.

    PubMed

    Gilbert, Sunny D; Stoddard, Colby D; Wise, Sarah J; Batey, Robert T

    2006-06-09

    Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.

  2. Copper(II) complexes with peptides based on the second cell binding site of fibronectin: metal coordination and ligand exchange kinetics.

    PubMed

    Pizzanelli, Silvia; Forte, Claudia; Pinzino, Calogero; Magrì, Antonio; La Mendola, Diego

    2016-02-07

    Copper(ii) complexes with short peptides based on the second cell binding site of fibronectin, PHSFN and PHSEN, have been characterized by potentiometric, UV-vis, CD, EPR and NMR spectroscopic methods. The histidine imidazole nitrogen is the anchoring site for the metal ion binding. Thermodynamic and spectroscopic evidence is given that the side chain oxygen donor atom of glutamyl residue in Ac-PHSEN-NH2 is also involved in the binding up to physiological pH. To determine ligand exchange kinetic parameters after the imidazole nitrogen anchoring, proton relaxation enhancement NMR data have been collected for the two hydrogen atoms of the imidazole ring in the temperature range 293-315 K at pH 5.2 and globally treated within different kinetic models for ligand exchange. The best fitting model involves two steps. In the first one, which is slow, a water molecule disengages a carbonyl or a carboxylate group coordinated to the metal ion in the complex formed by PHSFN or PHSEN, respectively. This stage is one order of magnitude slower for PHSEN, due to entropic effects. In the second step, which is fast, the complex just formed exchanges with the ligand. In this step, no appreciable differences are found for the two cases examined.

  3. Kinetic models for historical processes of fast invasion and aggression

    NASA Astrophysics Data System (ADS)

    Aristov, Vladimir V.; Ilyin, Oleg V.

    2015-04-01

    In the last few decades many investigations have been devoted to theoretical models in new areas concerning description of different biological, sociological, and historical processes. In the present paper we suggest a model of the Nazi Germany invasion of Poland, France, and the USSR based on kinetic theory. We simulate this process with the Cauchy boundary problem for two-element kinetic equations. The solution of the problem is given in the form of a traveling wave. The propagation velocity of a front line depends on the quotient between initial forces concentrations. Moreover it is obtained that the general solution of the model can be expressed in terms of quadratures and elementary functions. Finally it is shown that the front-line velocities agree with the historical data.

  4. Stochastic kinetics on networks: when slow is fast.

    PubMed

    Li, Xin; Kolomeisky, Anatoly B; Valleriani, Angelo

    2014-09-04

    Most chemical and biological processes can be viewed as reaction networks in which different pathways often compete kinetically for transformation of substrates into products. An enzymatic process is an example of such phenomena when biological catalysts create new routes for chemical reactions to proceed. It is typically assumed that the general process of product formation is governed by the pathway with the fastest kinetics at all time scales. In contrast to the expectation, here we show theoretically that at time scales sufficiently short, reactions are predominantly determined by the shortest pathway (in the number of intermediate states), regardless of the average turnover time associated with each pathway. This universal phenomenon is demonstrated by an explicit calculation for a system with two competing reversible (or irreversible) pathways. The time scales that characterize this regime and its relevance for single-molecule experimental studies are also discussed.

  5. Cyclam Derivatives with a Bis(phosphinate) or a Phosphinato-Phosphonate Pendant Arm: Ligands for Fast and Efficient Copper(II) Complexation for Nuclear Medical Applications.

    PubMed

    David, Tomáš; Kubíček, Vojtěch; Gutten, Ondrej; Lubal, Přemysl; Kotek, Jan; Pietzsch, Hans-Jürgen; Rulíšek, Lubomír; Hermann, Petr

    2015-12-21

    Cyclam derivatives bearing one geminal bis(phosphinic acid), -CH2PO2HCH2PO2H2 (H2L(1)), or phosphinic-phosphonic acid, -CH2PO2HCH2PO3H2 (H3L(2)), pendant arm were synthesized and studied as potential copper(II) chelators for nuclear medical applications. The ligands showed good selectivity for copper(II) over zinc(II) and nickel(II) ions (log KCuL = 25.8 and 27.7 for H2L(1) and H3L(2), respectively). Kinetic study revealed an unusual three-step complex formation mechanism. The initial equilibrium step leads to out-of-cage complexes with Cu(2+) bound by the phosphorus-containing pendant arm. These species quickly rearrange to an in-cage complex with cyclam conformation II, which isomerizes to another in-cage complex with cyclam conformation I. The first in-cage complex is quantitatively formed in seconds (pH ≈5, 25 °C, Cu:L = 1:1, cM ≈ 1 mM). At pH >12, I isomers undergo nitrogen atom inversion, leading to III isomers; the structure of the III-[Cu(HL(2))] complex in the solid state was confirmed by X-ray diffraction analysis. In an alkaline solution, interconversion of the I and III isomers is mutual, leading to the same equilibrium isomeric mixture; such behavior has been observed here for the first time for copper(II) complexes of cyclam derivatives. Quantum-chemical calculations showed small energetic differences between the isomeric complexes of H3L(2) compared with analogous data for isomeric complexes of cyclam derivatives with one or two methylphosphonic acid pendant arm(s). Acid-assisted dissociation proved the kinetic inertness of the complexes. Preliminary radiolabeling of H2L(1) and H3L(2) with (64)Cu was fast and efficient, even at room temperature, giving specific activities of around 70 GBq of (64)Cu per 1 μmol of the ligand (pH 6.2, 10 min, ca. 90 equiv of the ligand). These specific activities were much higher than those of H3nota and H4dota complexes prepared under identical conditions. The rare combination of simple ligand synthesis, very

  6. Bond formation kinetics affects self-assembly directed by ligand-receptor interactions.

    PubMed

    Jan Bachmann, Stephan; Petitzon, Marius; Mognetti, Bortolo Matteo

    2016-11-28

    In this paper we study aggregation kinetics in systems of particles functionalised by complementary linkers. Most of the coarse-grained models currently employed to study large-scale self-assembly of these systems rely on effective potentials between particles as calculated using equilibrium statistical mechanics. In these approaches the kinetic aspects underlying the formation of inter-particle linkages are neglected. We show how the rate at which supramolecular linkages form drastically changes the self-assembly pathway. In order to do this we develop a method that combines Brownian dynamics simulations with a Gillespie algorithm accounting for the evolution of inter-particle linkages. If compared with dynamics based on effective potentials, an explicit description of inter-particle linkages results in aggregates that in the early stages of self-assembly have a lower valency. Relaxation towards equilibrium is hampered by the time required to break existing linkages within one cluster and to reorient them toward free particles. This effect is more important at low temperature and high particle diffusion constant. Our results highlight the importance of including kinetic rates into coarse-grained descriptions of ligand-receptor systems.

  7. Carbon nanoparticle-modified multi-wall carbon nanotubes with fast adsorption kinetics for water treatment

    NASA Astrophysics Data System (ADS)

    Wang, Guan; Ren, Wei; Tan, Hui Ru; Liu, Ye

    2017-02-01

    Carbon nanoparticle-modified multi-wall carbon nanotubes were prepared using a dehydration of carbohydrate compound method. The structural change was characterized by transmission electron microscopy, Raman spectroscopy, and Brunauer, Emmett and Teller measurement. Fast adsorption kinetics was observed for multi-wall carbon nanotubes with modification, as demonstrated by the adsorption of the model compound methylene blue. This work provides a novel facile engineering strategy to equip multi-wall carbon nanotubes with fast adsorption kinetics, which is promising for efficient water purification.

  8. Carbon nanoparticle-modified multi-wall carbon nanotubes with fast adsorption kinetics for water treatment.

    PubMed

    Wang, Guan; Ren, Wei; Tan, Hui Ru; Liu, Ye

    2017-02-24

    Carbon nanoparticle-modified multi-wall carbon nanotubes were prepared using a dehydration of carbohydrate compound method. The structural change was characterized by transmission electron microscopy, Raman spectroscopy, and Brunauer, Emmett and Teller measurement. Fast adsorption kinetics was observed for multi-wall carbon nanotubes with modification, as demonstrated by the adsorption of the model compound methylene blue. This work provides a novel facile engineering strategy to equip multi-wall carbon nanotubes with fast adsorption kinetics, which is promising for efficient water purification.

  9. Kinetic theory of ligand recombination of myoglobin: a model for a combination of entropic and enthalpic effects

    NASA Astrophysics Data System (ADS)

    Yang, Dah-Yen; Sheu, Wen-Shyan; Sheu, Sheh-Yi; Lin, S. H.

    A kinetic theory of ligand recombination of myoglobin is obtained through a microscopic model. The macroscopic time dependent rate constant is obtained by the first passage time distribution random walk method. When the ligand is outside the haem pocket, it diffuses in a continuum space. In this process, this rate corresponds to a Smoluchowski rate constant times the concentration of myoglobin. After penetrating through the hydration shell, the ligand waits in front of the gate or diffuses on the myoglobin surface for entering the gate. This waiting time refers to a large scale fluctuation of protein to open the gate. When the ligand is inside the pocket, the motion of the ligand ranges from a ballistic to a diffusive limit. To cover the whole range of friction, it is necessary to solve exactly a finite area random walk model with periodic gating in one- and two-dimensional finite lattices with slippery boundary conditions. Protein dynamics influence the ligand motion indirectly through the collision between the ligand and the heme pocket well. The first step corresponds to an adiabatic dissociation process. A branching diagram method is used to show a detailed pathway analysis of the adiabaticity by introducing intermediate states in the quintet states for the CO ligand binding. The rate theories of ligand recombination of myoglobin are a combination of entropic and enthalpic effects.

  10. Role of water and steric constraints in the kinetics of cavity–ligand unbinding

    PubMed Central

    Tiwary, Pratyush; Mondal, Jagannath; Morrone, Joseph A.; Berne, B. J.

    2015-01-01

    A key factor influencing a drug’s efficacy is its residence time in the binding pocket of the host protein. Using atomistic computer simulation to predict this residence time and the associated dissociation process is a desirable but extremely difficult task due to the long timescales involved. This gets further complicated by the presence of biophysical factors such as steric and solvation effects. In this work, we perform molecular dynamics (MD) simulations of the unbinding of a popular prototypical hydrophobic cavity–ligand system using a metadynamics-based approach that allows direct assessment of kinetic pathways and parameters. When constrained to move in an axial manner, the unbinding time is found to be on the order of 4,000 s. In accordance with previous studies, we find that the cavity must pass through a region of sharp wetting transition manifested by sudden and high fluctuations in solvent density. When we remove the steric constraints on ligand, the unbinding happens predominantly by an alternate pathway, where the unbinding becomes 20 times faster, and the sharp wetting transition instead becomes continuous. We validate the unbinding timescales from metadynamics through a Poisson analysis, and by comparison through detailed balance to binding timescale estimates from unbiased MD. This work demonstrates that enhanced sampling can be used to perform explicit solvent MD studies at timescales previously unattainable, to our knowledge, obtaining direct and reliable pictures of the underlying physiochemical factors including free energies and rate constants. PMID:26371312

  11. Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase.

    PubMed

    Hothi, Parvinder; Basran, Jaswir; Sutcliffe, Michael J; Scrutton, Nigel S

    2003-04-08

    The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ

  12. Effect of ionic strength on ligand exchange kinetics between a mononuclear ferric citrate complex and siderophore desferrioxamine B

    NASA Astrophysics Data System (ADS)

    Ito, Hiroaki; Fujii, Manabu; Masago, Yoshifumi; Waite, T. David; Omura, Tatsuo

    2015-04-01

    The effect of ionic strength (I) on the ligand exchange reaction between a mononuclear ferric citrate complex and the siderophore, desferrioxamine B (DFB), was examined in the NaCl concentration range of 0.01-0.5 M, particularly focusing on the kinetics and mechanism of ligand exchange under environmentally relevant conditions. Overall ligand exchange rate constants were determined by spectrophotometrically measuring the time course of ferrioxamine B formation at a water temperature of 25 °C, pH 8.0, and citrate/Fe molar ratios of 500-5000. The overall ligand exchange rate decreased by 2-11-fold (depending on the citrate/Fe molar ratios) as I increased from approximately 0.01 to 0.5 M. In particular, a relatively large decrease was observed at lower I (<0.1 M). A ligand exchange model describing the effect of I on the ligand exchange rate via disjunctive and adjunctive pathways was developed by considering the pseudo-equilibration of ferric citrate complexes and subsequent ferrioxamine formation on the basis of the Eigen-Wilkins metal-ligand complexation theory. The model and experimental data consistently suggest that the adjunctive pathway (i.e., direct association of DFB with ferric mono- and di-citrate complexes following dissociation of citrate from the parent complexes) dominates in ferrioxamine formation under the experimental conditions used. The model also predicts that the higher rate of ligand exchange at lower I is associated with the decrease in the ferric dicitrate complex stability because of the relatively high electrical repulsion between ferric monocitrate and free citrate at lower I (note that the reactivity of ferric dicitrate with DFB is smaller than that for the monocitrate complex). Overall, the findings of this study contribute to the understanding of the potential effect of I on ligand exchange kinetics in natural waters and provide fundamental knowledge on iron transformation and bioavailability.

  13. Modulation of a ligand's energy landscape and kinetics by the chemical environment.

    PubMed

    Held, Martin; Imhof, Petra; Keller, Bettina G; Noé, Frank

    2012-11-26

    Understanding how the chemical environment modulates the predominant conformations and kinetics of flexible molecules is a core interest of biochemistry and a prerequisite for the rational design of synthetic catalysts. This study combines molecular dynamics simulation and Markov state models (MSMs) to a systematic computational strategy for investigating the effect of the chemical environment of a molecule on its conformations and kinetics. MSMs allow quantities to be computed that are otherwise difficult to access, such as the metastable sets, their free energies, and the relaxation time scales related to the rare transitions between metastable states. Additionally, MSMs are useful to identify observables that may act as sensors for the conformational or binding state of the molecule, thus guiding the design of experiments. In the present study, the conformation dynamics of UDP-GlcNAc are studied in vacuum, water, water + Mg(2+), and in the protein UDP-GlcNAc 2-epimerase. It is found that addition of Mg(2+) significantly affects the conformational stability, thermodynamics, and kinetics of UDP-GlcNAc. In particular, the slowest structural process, puckering of the GlcNAc sugar, depends on the overall conformation of UDP-GlcNAc and may thus act as a sensor of whether Mg(2+) is bound or not. Interestingly, transferring the molecule from vacuum to water makes the protein-binding conformations UDP-GlcNAc first accessible, while adding Mg(2+) further stabilizes them by specifically associating to binding-competent conformations. While Mg(2+) is not cocrystallized in the UDP-GlcNAc 2-epimerase complex, the selectively stabilized Mg(2+)/UDP-GlcNAc complex may be a template for the bound state, and Mg(2+) may accompany the binding-competent ligand conformation to the binding pocket. This serves as a possible explanation of the enhanced epimerization rate in the presence of Mg(2+). This role of Mg(2+) has previously not been described and opens the question whether

  14. Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.

    PubMed

    Sly, Krystal L; Conboy, John C

    2014-11-18

    Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 10(6) M(-1)·s(-1) at 0.43 μM PnA to (1.1 ± 0.1) × 10(5) M(-1)·s(-1) at 12 μM PnA. CTB-GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 10(9) M(-1)·s(-1) at 0.5 nM CTB to (3.5 ± 0.2) × 10(6) M(-1)·s(-1) at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 10(9) M(-1) and (2.8 ± 0.5) × 10(13) M(-1), respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB-GM1 demonstrates positive ligand-ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein-ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

  15. Kinetics and Mechanism of the Ligand Exchange Reaction Between Tetraaza Macrocycle Ligand and Cu(II) Tetradentate Amine-Amide Complexes.

    PubMed

    Vafazadeh, Rasoul; Zare-Sadrabadi, Ghasem

    2015-01-01

    The kinetics of the ligand exchange reaction of tetraaza macrocycle, teazma (teazmais 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-diene dihydrogen perchlorate) with Cu(bcen)(2+) and Cu(bctn)(2+), where bcen and bctn are N,N'-bis(β-carbamoylethyl) ethylendiamine) and N,N'-bis(β-carbamoylethyl) propylendiamine), respectively, have been studied by visible spectrophotometry in dimethylformamide, DMF, solvent at 25 ± 0.2°C. In the system of Cu(bctn)(2+)/teazma,the ligand exchange reaction proceeds in a two-step-consecutive manner, with two rate constants k(bctn)(obsd)(1) and k(bctn)(obsd)(2). The first reaction step was dependent on the concentration of teazma macrocycle, while the second reaction step was independent. However, it is found that the ligand exchange reaction in Cu(bcen)(2+)/teazma proceeds in an one-step with the rate constant k(bcen)(obsd). The rate constant is dependent on [teazma] macrocycle. The ligand exchange reaction in the system of Cu(bcen)(2+)/teazma is not complete and after some progress, the reaction reaches equilibrium. On the basis of results, a reaction mechanism is proposed and discussed for the ligand exchange rate.

  16. The pH dependence of heme pocket hydration and ligand rebinding kinetics in photodissociated carbonmonoxymyoglobin.

    PubMed

    Esquerra, Raymond M; Jensen, Russell A; Bhaskaran, Shyam; Pillsbury, Marlisa L; Mendoza, Juan L; Lintner, Benjamin W; Kliger, David S; Goldbeck, Robert A

    2008-05-16

    We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.

  17. The pH Dependence of Heme Pocket Hydration and Ligand Rebinding Kinetics in Photodissociated Carbonmonoxymyoglobin*

    PubMed Central

    Esquerra, Raymond M.; Jensen, Russell A.; Bhaskaran, Shyam; Pillsbury, Marlisa L.; Mendoza, Juan L.; Lintner, Benjamin W.; Kliger, David S.; Goldbeck, Robert A.

    2008-01-01

    We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation (Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A.103 ,1254 -125916432219). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change. PMID:18359768

  18. Simulation of the kinetics of ligand binding to a protein by molecular dynamics: geminate rebinding of nitric oxide to myoglobin.

    PubMed Central

    Schaad, O; Zhou, H X; Szabo, A; Eaton, W A; Henry, E R

    1993-01-01

    We have begun to use molecular dynamics to simulate the kinetics of nitric oxide rebinding to myoglobin after photodissociation. Rebinding was simulated using a potential function that switches smoothly between a nonbinding potential and a binding potential as a function of the position and orientation of the ligand, with no barrier arising from the crossing of potential surfaces of different electron spin. In 96 of 100 trajectories, the ligand rebound in < 15 ps. The kinetic progress curve was obtained by determining the time in each trajectory at which the ligand rebound and then calculating the fraction of unbound ligands as a function of time. The curve can be well reproduced by a simple model based on the dynamics of a Langevin particle moving on a one-dimensional potential of mean force calculated from nonreactive protein trajectories. The rate of escape from the energy well adjacent to the heme is in good agreement with the value calculated from experimental data, suggesting that a multiple-well model provides a plausible explanation for the nonexponential rebinding kinetics. A transition-state analysis suggests that protein conformational relaxation coupled to the displacement of the iron from the heme plane is an unlikely cause for the nonexponential rebinding of nitric oxide. PMID:8415739

  19. The use of kinetic modelling as a fast way to screen thermal endurance of space materials

    NASA Astrophysics Data System (ADS)

    Moser, M.; Heltzel, S.; Semprimoschnig, C.; Garcia Martin, G.

    2003-09-01

    Currently planned missions of ESA (European Space Agency) to the inner part of the solar system will require the use of materials at an extreme radiation and temperature environment. A major concern regarding the selection of these materials is the thermal behaviour and the thermal stability. In this paper two kinetic models, the one following the ASTM E 1641 and ASTM E 1877 standards and the other following the Model Free Kinetics (MFK) approach, are presented. These models allow an easy and fast way to screen the thermal endurance of organic materials by running Thermo Gravimetric Analyses (TGA) temperature scans.

  20. Ultra-fast dynamic compression technique to study kinetics of phase transformations in Bismuth

    SciTech Connect

    Smith, R F; Kane, J O; Eggert, J H; Saculla, M D; Jankowski, A F; Bastea, M; Hicks, D G; Collins, G W

    2007-12-28

    Pre-heated Bi was ramp compressed within 30 ns to a peak stress of {approx}11 GPa to explore structural phase transformation kinetics under dynamic loading conditions. Under these ultra-fast compression time-scales the equilibrium Bi I-II phase boundary is overpressurized by {Delta}P {approx} 0.8 GPa. {Delta}P is observed to increase logarithmically with strain rate, {var_epsilon}, above 10{sup 6} s{sup -1}. Estimates from a kinetics model predict that the Bi I phase is fully transformed within 3 ns.

  1. Different epidermal growth factor (EGF) receptor ligands show distinct kinetics and biased or partial agonism for homodimer and heterodimer formation.

    PubMed

    Macdonald-Obermann, Jennifer L; Pike, Linda J

    2014-09-19

    The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics.

    PubMed

    Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A; Arvizu-Flores, Aldo A; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A; García-Orozco, Karina D; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R; Islas-Osuna, Maria A

    2017-04-01

    We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM min(-1) and 68.49 s(-1) respectively and 0.693 mM, 105.32 mM min(-1) and 89.57 s(-1), for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.

  3. Kinetics of protein-ligand unbinding via smoothed potential molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Mollica, Luca; Decherchi, Sergio; Zia, Syeda Rehana; Gaspari, Roberto; Cavalli, Andrea; Rocchia, Walter

    2015-06-01

    Drug discovery is expensive and high-risk. Its main reasons of failure are lack of efficacy and toxicity of a drug candidate. Binding affinity for the biological target has been usually considered one of the most relevant figures of merit to judge a drug candidate along with bioavailability, selectivity and metabolic properties, which could depend on off-target interactions. Nevertheless, affinity does not always satisfactorily correlate with in vivo drug efficacy. It is indeed becoming increasingly evident that the time a drug spends in contact with its target (aka residence time) can be a more reliable figure of merit. Experimental kinetic measurements are operatively limited by the cost and the time needed to synthesize compounds to be tested, to express and purify the target, and to setup the assays. We present here a simple and efficient molecular-dynamics-based computational approach to prioritize compounds according to their residence time. We devised a multiple-replica scaled molecular dynamics protocol with suitably defined harmonic restraints to accelerate the unbinding events while preserving the native fold. Ligands are ranked according to the mean observed scaled unbinding time. The approach, trivially parallel and easily implementable, was validated against experimental information available on biological systems of pharmacological relevance.

  4. Computational fluid dynamics modelling of biomass fast pyrolysis in fluidised bed reactors, focusing different kinetic schemes.

    PubMed

    Ranganathan, Panneerselvam; Gu, Sai

    2016-08-01

    The present work concerns with CFD modelling of biomass fast pyrolysis in a fluidised bed reactor. Initially, a study was conducted to understand the hydrodynamics of the fluidised bed reactor by investigating the particle density and size, and gas velocity effect. With the basic understanding of hydrodynamics, the study was further extended to investigate the different kinetic schemes for biomass fast pyrolysis process. The Eulerian-Eulerian approach was used to model the complex multiphase flows in the reactor. The yield of the products from the simulation was compared with the experimental data. A good comparison was obtained between the literature results and CFD simulation. It is also found that CFD prediction with the advanced kinetic scheme is better when compared to other schemes. With the confidence obtained from the CFD models, a parametric study was carried out to study the effect of biomass particle type and size and temperature on the yield of the products.

  5. Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

    PubMed Central

    2009-01-01

    Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

  6. Ligand-specific interactions modulate kinetic, energetic and mechanical properties of the human β2 adrenergic receptor

    PubMed Central

    Zocher, Michael; Fung, Juan J; Kobilka, Brian K; Müller, Daniel J

    2015-01-01

    SUMMARY G-protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remains to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand-binding modulates the energy landscape of the human β2 adrenergic receptor (β2AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic and mechanical properties of functionally important structural regions of β2AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns. PMID:22748765

  7. Synthesis, characterization, dynamics and reactivity toward amination of η3-allyl palladium complexes bearing mixed ancillary ligands. Evaluation of the electronic characteristics of the ligands from kinetic data.

    PubMed

    Canovese, Luciano; Visentin, Fabiano; Levi, Carlo; Dolmella, Alessandro

    2011-01-28

    On the basis of an original protocol, we have synthesized several complexes of the type [Pd(η(3)-C(3)H(3)R(2))(LL')]ClO(4) (R = H, Me; L, L' = PPh(3), P(OEt)(3), 2,6-dimethylphenylisocyanide, t-butylisocyanide, 1,3-dimesitylimidazolidine, 1,3-dimesitylimidazol-2-ylidene). The complexes, some of which are completely new species, were fully characterized and their behaviour in solution was studied by means of (1)H NMR. The reactions of the complexes bearing the symmetric allyl moiety [Pd(η(3)-C(3)H(5))(LL')]ClO(4) with piperidine in the presence of the olefin dimethylfumarate were followed under kinetically controlled conditions. Formation of allyl-amine and of the palladium(0) derivatives [Pd(η(2)-dmfu)(LL'] was observed. The reaction rates k(2) proved to be strongly dependent on the ancillary ligand nature and allowed a direct comparison among the electronic characteristics of the ligands. The reactivity trend determined appears to be mainly influenced by the capability of the ancillary ligands in transferring electron density to the metal centre and consequently on the allyl fragment.

  8. Validation of an empirical RNA-ligand scoring function for fast flexible docking using RiboDock®

    NASA Astrophysics Data System (ADS)

    Morley, S. David; Afshar, Mohammad

    2004-03-01

    We report the design and validation of a fast empirical function for scoring RNA-ligand interactions, and describe its implementation within RiboDock®, a virtual screening system for automated flexible docking. Building on well-known protein-ligand scoring function foundations, features were added to describe the interactions of common RNA-binding functional groups that were not handled adequately by conventional terms, to disfavour non-complementary polar contacts, and to control non-specific charged interactions. The results of validation experiments against known structures of RNA-ligand complexes compare favourably with previously reported methods. Binding modes were well predicted in most cases and good discrimination was achieved between native and non-native ligands for each binding site, and between native and non-native binding sites for each ligand. Further evidence of the ability of the method to identify true RNA binders is provided by compound selection (`enrichment factor') experiments based around a series of HIV-1 TAR RNA-binding ligands. Significant enrichment in true binders was achieved amongst high scoring docking hits, even when selection was from a library of structurally related, positively charged molecules. Coupled with a semi-automated cavity detection algorithm for identification of putative ligand binding sites, also described here, the method is suitable for the screening of very large databases of molecules against RNA and RNA-protein interfaces, such as those presented by the bacterial ribosome. Abbreviations: ACD - Available Chemicals Directory; AMP - adenosine monophosphate; EF - enrichment factor; FMN - flavin mononucleotide; FRET - fluorescence resonance energy transfer; RMSD - root mean square deviation; TAR - trans-activation response element; Tat - transcriptional activator protein.

  9. Nanosecond Pulse Discharges and Fast Ionization Wave Discharges: Fundamental Kinetic Processes and Applications

    NASA Astrophysics Data System (ADS)

    Adamovich, Igor

    2011-10-01

    Over the last two decades, nanosecond pulse discharges and Fast Ionization Wave (FIW) discharges have been studied extensively, both theoretically and experimentally. Current interest in characterization of these discharges is driven mainly by their potential for applications such as plasma chemical fuel reforming, plasma-assisted combustion, high-speed flow control, pumping of electric discharge excited lasers, and generation of high-energy electrons. A unique capability of FIW discharges to generate significant ionization and high concentrations of excited species at high pressures and over large distances, before ionization instabilities have time to develop, is very attractive for these applications. Recent advances in laser optical diagnostics offer an opportunity of making non-intrusive, spatially and time-resolved measurements of electron density and electric field distributions in high-speed ionization wave discharges, on nanosecond time scale. Insight into FIW formation and propagation dynamics also requires development of predictive kinetic models, and their experimental validation. Although numerical kinetic models may incorporate detailed kinetics of charged and neutral species in the propagating ionization wave front (including non-local electron kinetics), analytic models are also attractive due to their capability of elucidating fundamental trends of discharge development. The talk gives an overview of recent progress in experimental characterization and kinetic modeling of nanosecond pulse and fast ionization wave discharges in nitrogen and air over a wide range of pulse repetition rates, 0.1-40 kHz. FIW discharge plasmas sustained at high pulse repetition rates are diffuse and volume filling, with most of the power coupled to the plasma behind the wave, at E/N = 200-300 Td and energy loading of 1-2 meV/molecule/pulse. The results demonstrate significant potential of large volume, diffuse, high pulse repetition rate FIW discharges for novel plasma

  10. Gyro-Kinetic Electron and Fully-Kinetic Ion Simulations of Fast Magnetosonic Waves in the Magnetosphere

    NASA Astrophysics Data System (ADS)

    Gao, X.; Liu, K.; Wang, X.; Min, K.; Lin, Y.

    2016-12-01

    Two-dimensional simulations using a gyro-kinetic electron and fully-kinetic ion (GeFi) scheme are preformed to study the excitation of fast magnetosonic waves in the magnetosphere, which arise from the ion Bernstein instability driven by ring-like proton velocity distributions (with a positive slope with respect to the perpendicular velocity). Since both ion and electron kinetics are relevant, particle-in-cell (PIC) simulations have often been employed to study the wave excitation. However, such simulations are limited to reduced ion-to-electron mass ratio (mi/me) and light-to-Alfvén speed ratio (c/VA) due to the computationally expensive nature of PIC codes. The present study exploits a GeFi scheme that can break through these limitations and use larger/more realistic mi/me and c/VA. The capability of the GeFi code in simulating the ion Bernstein instability is first demonstrated by comparing a GeFi simulation using reduced mass ratio (mi/me=100) and speed ratio (c/VA=15) to a corresponding PIC simulation. A realistic speed ratio (c/VA=400) and a larger mass ratio (mi/me=400) are then adopted in the GeFi code to explore how the results vary. It is shown that the increased mi/me and c/VA lead to a larger lower hybrid frequency and allow waves to arise at more ion cyclotron harmonics, consistent with the general prediction of linear dispersion theory.

  11. Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics.

    PubMed

    Bingaman, Jamie L; Messina, Kyle J; Bevilacqua, Philip C

    2017-03-14

    Reaction kinetics on the millisecond timescale pervade the protein and RNA fields. To study such reactions, investigators often perturb the system with abiological solution conditions or substrates in order to slow the rate to timescales accessible by hand mixing; however, such perturbations can change the rate-limiting step and obscure key folding and chemical steps that are found under biological conditions. Mechanical methods for collecting data on the millisecond timescale, which allow these perturbations to be avoided, have been developed over the last few decades. These methods are relatively simple and can be conducted on affordable and commercially available instruments. Here, we focus on using the rapid quench-flow technique to study the fast reaction kinetics of RNA enzymes, or ribozymes, which often react on the millisecond timescale under biological conditions. Rapid quench of ribozymes is completely parallel to the familiar hand-mixing approach, including the use of radiolabeled RNAs and fractionation of reactions on polyacrylamide gels. We provide tips on addressing and preventing common problems that can arise with the rapid-quench technique. Guidance is also offered on ensuring the ribozyme is properly folded and fast-reacting. We hope that this article will facilitate the broader use of rapid-quench instrumentation to study fast-reacting ribozymes under biological reaction conditions.

  12. Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics.

    PubMed

    Pujol-Vila, F; Vigués, N; Díaz-González, M; Muñoz-Berbel, X; Mas, J

    2015-05-15

    Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.

  13. Effect of carboxylic and thiol ligands (oxalate, cysteine) on the kinetics of desorption of Hg(II) from kaolinite

    SciTech Connect

    Senevirathna, W. U.; Zhang, Hong; Gu, Baohua

    2010-01-01

    Sorption and desorption of Hg(II) on clay minerals can impact the biogeochemical cycle and bio-uptake of Hg in the environment. We studied the kinetics of the desorption of Hg(II) from kaolinite as affected by oxalate and cysteine, representing the ligands with carboxylic and thiol groups of different affinities for Hg(II). The effects of pH (3, 5, and 7), ligand concentration (0.25 and 1.0 mM), and temperature (15 C, 25 C, and 35 C) on the Hg(II) desorption were investigated through desorption kinetics. Our study showed that the Hg(II) desorption was pH dependent. In the absence of any organic ligand, >90% of the previously adsorbed Hg(II) desorbed at pH 3 within 2 h, compared to <10% at pH 7. Similar results were observed in the presence of oxalate, showing that it hardly affected the Hg(II) desorption. Cysteine inhibited the Hg(II) desorption significantly at all the pH tested, especially in the first 80 min with the desorption less than 20%, but the inhibition of the desorption appeared to be less prominent afterwards. The effect of the ligand concentration on the Hg(II) desorption was small, especially in the presence of oxalate. The effect of temperature on the Hg(II) desorption was nearly insignificant. The effect of the organic acids on the Hg(II) sorption and desorption is explained by the formation of the ternary surface complexes involving the mineral, ligand, and Hg(II). The competition for Hg(II) between the cysteine molecules adsorbed on the particle surfaces and in the solution phase probably can also affect the Hg(II) desorption.

  14. A fast-start pacing strategy speeds pulmonary oxygen uptake kinetics and improves supramaximal running performance.

    PubMed

    Turnes, Tiago; Salvador, Amadeo Félix; Lisbôa, Felipe Domingos; de Aguiar, Rafael Alves; Cruz, Rogério Santos de Oliveira; Caputo, Fabrizio

    2014-01-01

    The focus of the present study was to investigate the effects of a fast-start pacing strategy on running performance and pulmonary oxygen uptake (VO2) kinetics at the upper boundary of the severe-intensity domain. Eleven active male participants (28±10 years, 70±5 kg, 176±6 cm, 57±4 mL/kg/min) visited the laboratory for a series of tests that were performed until exhaustion: 1) an incremental test; 2) three laboratory test sessions performed at 95, 100 and 110% of the maximal aerobic speed; 3) two to four constant speed tests for the determination of the highest constant speed (HS) that still allowed achieving maximal oxygen uptake; and 4) an exercise based on the HS using a higher initial speed followed by a subsequent decrease. To predict equalized performance values for the constant pace, the relationship between time and distance/speed through log-log modelling was used. When a fast-start was utilized, subjects were able to cover a greater distance in a performance of similar duration in comparison with a constant-pace performance (constant pace: 670 m±22%; fast-start: 683 m±22%; P = 0.029); subjects also demonstrated a higher exercise tolerance at a similar average speed when compared with constant-pace performance (constant pace: 114 s±30%; fast-start: 125 s±26%; P = 0.037). Moreover, the mean VO2 response time was reduced after a fast start (constant pace: 22.2 s±28%; fast-start: 19.3 s±29%; P = 0.025). In conclusion, middle-distance running performances with a duration of 2-3 min are improved and VO2 response time is faster when a fast-start is adopted.

  15. A Fast-Start Pacing Strategy Speeds Pulmonary Oxygen Uptake Kinetics and Improves Supramaximal Running Performance

    PubMed Central

    Turnes, Tiago; Salvador, Amadeo Félix; Lisbôa, Felipe Domingos; de Aguiar, Rafael Alves; Cruz, Rogério Santos de Oliveira; Caputo, Fabrizio

    2014-01-01

    The focus of the present study was to investigate the effects of a fast-start pacing strategy on running performance and pulmonary oxygen uptake () kinetics at the upper boundary of the severe-intensity domain. Eleven active male participants (28±10 years, 70±5 kg, 176±6 cm, 57±4 mL/kg/min) visited the laboratory for a series of tests that were performed until exhaustion: 1) an incremental test; 2) three laboratory test sessions performed at 95, 100 and 110% of the maximal aerobic speed; 3) two to four constant speed tests for the determination of the highest constant speed (HS) that still allowed achieving maximal oxygen uptake; and 4) an exercise based on the HS using a higher initial speed followed by a subsequent decrease. To predict equalized performance values for the constant pace, the relationship between time and distance/speed through log-log modelling was used. When a fast-start was utilized, subjects were able to cover a greater distance in a performance of similar duration in comparison with a constant-pace performance (constant pace: 670 m±22%; fast-start: 683 m±22%; P = 0.029); subjects also demonstrated a higher exercise tolerance at a similar average speed when compared with constant-pace performance (constant pace: 114 s±30%; fast-start: 125 s±26%; P = 0.037). Moreover, the mean response time was reduced after a fast start (constant pace: 22.2 s±28%; fast-start: 19.3 s±29%; P = 0.025). In conclusion, middle-distance running performances with a duration of 2–3 min are improved and response time is faster when a fast-start is adopted. PMID:25360744

  16. Fast electron energy deposition in a magnetized plasma: Kinetic theory and particle-in-cell simulation

    SciTech Connect

    Robiche, J.; Rax, J.-M.; Bonnaud, G.; Gremillet, L.

    2010-03-15

    The collisional dynamics of a relativistic electron jet in a magnetized plasma are investigated within the framework of kinetic theory. The relativistic Fokker-Planck equation describing slowing down, pitch angle scattering, and cyclotron rotation is derived and solved. Based on the solution of this Fokker-Planck equation, an analytical formula for the root mean square spot size transverse to the magnetic field is derived and this result predicts a reduction in radial transport. Some comparisons with particle-in-cell simulation are made and confirm striking agreement between the theory and the simulation. For fast electron with 1 MeV typical kinetic energy interacting with a solid density hydrogen plasma, the energy deposition density in the transverse direction increases by a factor 2 for magnetic field of the order of 1 T. Along the magnetic field, the energy deposition profile is unaltered compared with the field-free case.

  17. Kinetics of Hg(II) exchange between organic ligands, goethite, and natural organic matter studied with an enriched stable isotope approach.

    PubMed

    Jiskra, Martin; Saile, Damian; Wiederhold, Jan G; Bourdon, Bernard; Björn, Erik; Kretzschmar, Ruben

    2014-11-18

    The mobility and bioavailability of toxic Hg(II) in the environment strongly depends on its interactions with natural organic matter (NOM) and mineral surfaces. Using an enriched stable isotope approach, we investigated the exchange of Hg(II) between dissolved species (inorganically complexed or cysteine-, EDTA-, or NOM-bound) and solid-bound Hg(II) (carboxyl-/thiol-resin or goethite) over 30 days under constant conditions (pH, Hg and ligand concentrations). The Hg(II)-exchange was initially fast, followed by a slower phase, and depended on the properties of the dissolved ligands and sorbents. The results were described by a kinetic model allowing the simultaneous determination of adsorption and desorption rate coefficients. The time scales required to reach equilibrium with the carboxyl-resin varied greatly from 1.2 days for Hg(OH)2 to 16 days for Hg(II)-cysteine complexes and approximately 250 days for EDTA-bound Hg(II). Other experiments could not be described by an equilibrium model, suggesting that a significant fraction of total-bound Hg was present in a non-exchangeable form (thiol-resin and NOM: 53-58%; goethite: 22-29%). Based on the slow and incomplete exchange of Hg(II) described in this study, we suggest that kinetic effects must be considered to a greater extent in the assessment of the fate of Hg in the environment and the design of experimental studies, for example, for stability constant determination or metal isotope fractionation during sorption.

  18. Method of experimental determination of the kinetic constants in fast polymerization reactions in nonisothermal diffusion conditions

    SciTech Connect

    Prochukhan, Yu.A.; Berlin, A.A.; Enikolopyan, N.S.

    1986-09-01

    A new method for the experimental determination of the kinetic constants k/sub p/ and k/sub t/ in fast polymerization reactions on the example of cationic (under the effect of AlCl/sub 3/, BF/sub 3/, and other catalysts) liquid phase polymerization of isobutylene in a flow was suggested. The study of the macrokinetic features of low-temperature polymerization of isobutylene revealed the specific conditions of the occurrence of the process (quasi-ideal displacement) which are characterized by the relative constancy and uniformity of the distribution of the concentrations of the reacting substances along the flow section.

  19. Protein-ligand recognition using spherical harmonic molecular surfaces: towards a fast and efficient filter for large virtual throughput screening.

    PubMed

    Cai, Wensheng; Shao, Xueguang; Maigret, Bernard

    2002-01-01

    Molecular surfaces are important because surface-shape complementarity is often a necessary condition in protein-ligand interactions and docking studies. We have previously described a fast and efficient method to obtain triangulated surface-meshes by topologically mapping ellipsoids on molecular surfaces. In this paper, we present an extension of our work to spherical harmonic surfaces in order to approximate molecular surfaces of both ligands and receptor-cavities and to easily check the surface-shape complementarity. The method consists of (1) finding lobes and holes on both ligand and cavity surfaces using contour maps of radius functions with spherical harmonic expansions, (2) superposing the surfaces around a given binding site by minimizing the distance between their respective expansion coefficients. This docking procedure capabilities was demonstrated by application to 35 protein-ligand complexes of known crystal structures. The method can also be easily and efficiently used as a filter to detect in a large conformational sampling the possible conformations presenting good complementarity with the receptor site, and being, therefore, good candidates for further more elaborate docking studies. This "virtual screening" was demonstrated on the platelet thrombin receptor.

  20. Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

    PubMed Central

    Di Iorio, E E; Winterhalter, K H; Giacometti, G M

    1987-01-01

    The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix. PMID:3567310

  1. 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

  2. Fast force field-based optimization of protein-ligand complexes with graphics processor.

    PubMed

    Heinzerling, Lennart; Klein, Robert; Rarey, Matthias

    2012-12-15

    Usually based on molecular mechanics force fields, the post-optimization of ligand poses is typically the most time-consuming step in protein-ligand docking procedures. In return, it bears the potential to overcome the limitations of discretized conformation models. Because of the parallel nature of the problem, recent graphics processing units (GPUs) can be applied to address this dilemma. We present a novel algorithmic approach for parallelizing and thus massively speeding up protein-ligand complex optimizations with GPUs. The method, customized to pose-optimization, performs at least 100 times faster than widely used CPU-based optimization tools. An improvement in Root-Mean-Square Distance (RMSD) compared to the original docking pose of up to 42% can be achieved. Copyright © 2012 Wiley Periodicals, Inc.

  3. Fast pyrolysis kinetics of alkali lignin: Evaluation of apparent rate parameters and product time evolution.

    PubMed

    Ojha, Deepak Kumar; Viju, Daniel; Vinu, R

    2017-10-01

    In this study, the apparent kinetics of fast pyrolysis of alkali lignin was evaluated by obtaining isothermal mass loss data in the timescale of 2-30s at 400-700°C in an analytical pyrolyzer. The data were analyzed using different reaction models to determine the rate constants and apparent rate parameters. First order and one dimensional diffusion models resulted in good fits with experimental data with apparent activation energy of 23kJmol(-1). Kinetic compensation effect was established using a large number of kinetic parameters reported in the literature for pyrolysis of different lignins. The time evolution of the major functional groups in the pyrolysate was analyzed using in situ Fourier transform infrared spectroscopy. Maximum production of the volatiles occurred around 10-12s. A clear transformation of guaiacols to phenol, catechol and their derivatives, and aromatic hydrocarbons was observed with increasing temperature. The plausible reaction steps involved in various transformations are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. A simple and fast kinetic assay for phytases using phytic acid-protein complex as substrate.

    PubMed

    Tran, Thuy Thi; Hatti-Kaul, Rajni; Dalsgaard, Søren; Yu, Shukun

    2011-03-15

    Phytase (EC 3.1.3.-) hydrolyzes phytate (IP(6)) present in cereals and grains to release inorganic phosphate (P(i)), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the P(i) liberated from IP(6). This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP(6)-lysozyme as a substrate. The assay is based on the principle that IP(6) forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP(6) in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released P(i) from IP(6). This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl(2), and phosphate, showed a concentration-dependent interference.

  5. Kinetic evidence for the existence of a rate-limiting step in the reaction of ferric hemoproteins with anionic ligands.

    PubMed

    Coletta, M; Angeletti, M; De Sanctis, G; Cerroni, L; Giardina, B; Amiconi, G; Ascenzi, P

    1996-01-15

    The kinetics of azide and fluroide binding to various monomeric and tetrameric ferric hemoproteins (sperm whale Mb, isolated alpha and beta chains of human Hb reacted with p-chloromercuribenzoate, dromeday, ox and human Hb) has been investigated (at pH 6.5 and 20 degrees C over a large range (20 microM to 2 M) of ligand concentration. It has been observed that the pseuo-first-order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptomatic concentration-independent value typical for each hemoprotein. This behavior, which has been detected only by an investigation covering an unusually large range of ligand concentrations appears to be independent of the ionic strength, and it underlies the existence of a rate-limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo- first-order rate constant becomes faster than a given value characteristic of the specific hemoprotein. Such a behavior is not observed in the case of fluroide binding probably because the pesudo- first-order rate constant for this ligand is much slower and never attains a value faster than that of the rate-limiting step. In general terms, this feature should involve a conformational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into the heme pocket and its reaction with the ferric iron.

  6. Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat.

    PubMed

    Andruchov, Oleg; Galler, Stefan

    2008-03-01

    This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

  7. High-speed gas chromatography in doping control: fast-GC and fast-GC/MS determination of beta-adrenoceptor ligands and diuretics.

    PubMed

    Brunelli, Claudio; Bicchi, Carlo; Di Stilo, Antonella; Salomone, Alberto; Vincenti, Marco

    2006-12-01

    In official doping controls, about 300 drugs and metabolites have to be screened for each sample. Moreover, the number of determinations to be routinely processed increases continuously as the number of both samples and potential illicit drugs keeps growing. As a consequence, increasingly specific, sensitive, and, above all, fast methods for doping controls are needed. The present study presents an efficient fast-GC/MS approach to the routine screening of two different classes of doping agents, namely beta-adrenoceptor ligands and diuretics (belonging to the S3, P2, and S5 groups of the WADA list of prohibited substances). Narrow bore columns (100 mm id) of different lengths and coated with apolar stationary phases were successfully used to separate the derivatized analytes; preliminary experiments (results not shown) showed better performances with OV-1701 for the separation of beta-adrenoceptor ligands. On the same stationary phase some diuretics required too high a temperature or a long isothermal time for elution, in which case a DB1-MS column was preferred. Two methods of sample preparation, derivatization, and analysis were used on aqueous standard mixtures of, respectively, (i) eight beta-adrenoceptor ligands, including five beta-antagonists (acebutolol, alprenolol, atenolol, metoprolol, pindolol) and three beta2-agonists (salbutamol, clenbuterol, terbutaline) and (ii) seventeen diuretic drugs (acetazolamide, althiazide, bendroflumethiazide, bumethanide, canrenone, chlorothiazide, chlortalidone, clopamide, ethacrinic acid, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, indomethacine, spironolactone, triamterene, trichloromethiazide) and one masking agent (probenecid). The mixture of beta-adrenoceptor ligand derivatives was efficiently separated in about 5.6 min, while the one of 18 diuretics and masking agents required less than 5 min for analysis. Limits of detection were from 1 microg/L for pindolol, ethacrinic acid, furosemide

  8. Kinetically stable metal ligand charge transfer complexes as crosslinks in nanogels/hydrogels: Physical properties and cytotoxicity.

    PubMed

    Wang, Rong; Both, Sanne K; Geven, Mike; Calucci, Lucia; Forte, Claudia; Dijkstra, Pieter J; Karperien, Marcel

    2015-10-01

    kinetically stable complexes are not cytotoxic even when embedded in hydrogels. These results provide fundamental issues to be considered in the design of hydrogels crosslinked through metal ligand complexation. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  9. Bifunctional cyclam-based ligands with phosphorus acid pendant moieties for radiocopper separation: thermodynamic and kinetic studies.

    PubMed

    Paúrová, Monika; Havlíčková, Jana; Pospíšilová, Aneta; Vetrík, Miroslav; Císařová, Ivana; Stephan, Holger; Pietzsch, Hans-Jürgen; Hrubý, Martin; Hermann, Petr; Kotek, Jan

    2015-03-16

    Two macrocyclic ligands based on cyclam with trans-disposed N-methyl and N-(4-aminobenzyl) substituents as well as two methylphosphinic (H2L1) or methylphosphonic (H4L2) acid pendant arms were synthesised and investigated in solution. The ligands form stable complexes with transition metal ions. Both ligands show high thermodynamic selectivity for divalent copper over nickel(II) and zinc(II)-K(CuL) is larger than K(Ni/ZnL) by about seven orders of magnitude. Complexation is significantly faster for the phosphonate ligand H4L2, probably due to the stronger coordination ability of the more basic phosphonate groups, which efficiently bind the metal ion in an "out-of-cage" complex and thus accelerate its "in-cage" binding. The rate of Cu(II) complexation by the phosphinate ligand H2L1 is comparable to that of cyclam itself and its derivatives with non-coordinating substituents. Acid-assisted decomplexation of the copper(II) complexes is relatively fast (τ1/2 = 44 and 42 s in 1 M aq. HClO4 at 25 °C for H2L1 and H4L2, respectively). This combination of properties is convenient for selective copper removal/purification. Thus, the title ligands were employed in the preparation of ion-selective resins for radiocopper(II) separation. Glycidyl methacrylate copolymer beads were modified with the ligands through a diazotisation reaction. The separation ability of the modified polymers was tested with cold copper(II) and non-carrier-added (64)Cu in the presence of a large excess of both nickel(II) and zinc(II). The experiments exhibited high overall separation efficiency leading to 60-70% recovery of radiocopper with high selectivity over the other metal ions, which were originally present in 900-fold molar excess. The results showed that chelating resins with properly tuned selectivity of their complexing moieties can be employed for radiocopper separation.

  10. On the Upscaling of Reaction-Transport Processes in Porous Media with Fast Kinetics

    SciTech Connect

    Kechagi, P.; Tsimpanogiannis, I.; Yortsos, Y.C.; Lichtner, P.

    2001-01-09

    This report is organized as follows: Provide a brief review of the upscaling constraints of the type (2) for a typical diffusion-reaction system. In this an analogy with two-phase flow in porous media was drawn. Then, using the methodology of QW a problem at the unit cell for the computation of the effective mass transfer coefficient, in processes where local thermodynamic equilibrium applies was derived. This problem is found to be different than in QW, as it depends on the gradients of the macroscale variable, and can be cast in terms of an eigenvalue problem. Two simple, examples, one involving advection-dissolution and another involving drying in a pore network, was presented to illustrate the coupling between scales and to show the quantitative effect in case this coupling was neglected. Finally, similar ideas and an illustrative example was applied to reaction-diffusion systems with fast kinetics, where an equilibrium state is approached.

  11. Chronic impact of sulfamethoxazole on acetate utilization kinetics and population dynamics of fast growing microbial culture.

    PubMed

    Kor-Bicakci, G; Pala-Ozkok, I; Rehman, A; Jonas, D; Ubay-Cokgor, E; Orhon, D

    2014-08-01

    The study evaluated the chronic impact of sulfamethoxazole on metabolic activities of fast growing microbial culture. It focused on changes induced on utilization kinetics of acetate and composition of the microbial community. The experiments involved a fill and draw reactor, fed with acetate and continuous sulfamethoxazole dosing of 50 mg/L. The evaluation relied on model evaluation of the oxygen uptake rate profiles, with parallel assessment of microbial community structure by 454-pyrosequencing. Continuous sulfamethoxazole dosing inflicted a retardation effect on acetate utilization in a way commonly interpreted as competitive inhibition, blocked substrate storage and accelerated endogenous respiration. A fraction of acetate was utilized at a much lower rate with partial biodegradation of sulfamethoxazole. Results of pyrosequencing with a replacement mechanism within a richer more diversified microbial culture, through inactivation of vulnerable fractions in favor of species resistant to antibiotic, which made them capable of surviving and competing even with a slower metabolic response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Lipoproteins attenuate TLR2 and TLR4 activation by bacteria and bacterial ligands with differences in affinity and kinetics.

    PubMed

    van Bergenhenegouwen, Jeroen; Kraneveld, Aletta D; Rutten, Lieke; Garssen, Johan; Vos, Arjan P; Hartog, Anita

    2016-10-28

    The small intestine is a specialized compartment were close interactions take place between host, microbes, food antigens and dietary fatty acids. Dietary fats get absorbed by epithelial cells and processed into a range of lipoprotein particles after which they are basolaterally secreted and collected in the lymphatics. In contrast to the colon, the small intestine is covered only by a thin mucus coat that allows for intimate interactions between host-cells and microbes. Lipoproteins have long been recognized as protective factors in infectious diseases via the neutralization of bacterial toxins like lipopolysaccharides. Much less attention has been given to the potential role of lipoproteins as factors contributing to the maintenance of small intestinal immune homeostasis via modulating bacteria-induced immune responses. Lipoproteins VLDL, LDL and HDL were found to neutralize TLR responses towards specific TLR-ligands or a selection of gram-negative and gram-positive bacteria. Attenuation of TLR2 activity was acute and only slightly improved by longer pre-incubation times of ligands and lipoproteins with no differences between bacterial-lipopeptides or bacteria. In contrast, attenuation of TLR4 responses was only observed after extensive preincubation of lipoproteins and LPS. Preincubation of bacteria and lipoproteins led only to a modest attenuation of TLR4 activity. Moreover, compared to TLR2, TLR4 activity could only be attenuated by lipoproteins over a small ligand dose range. These results demonstrate the ability of lipoproteins VLDL, LDL and HDL to inhibit TLR responses towards bacterial-ligands and bacteria. Presence of lipoproteins was found to modulate the MAMP-induced cytokine release by primary human monocytes measured as changes in the release of IL-6, TNFα, GM-CSF and IFNγ. Using TLR2 and TLR4-reporter cells, lipoproteins were found to inhibit TLR responses with differences in affinity and kinetics. These data establish a role for lipoproteins as

  13. Ligand-interaction kinetics of the pheromone- binding protein from the gypsy moth, L. dispar: insights into the mechanism of binding and release.

    PubMed

    Gong, Yongmei; Pace, Tamara C S; Castillo, Carlos; Bohne, Cornelia; O'Neill, Melanie A; Plettner, Erika

    2009-02-27

    The pheromone-binding proteins (PBPs), which exist at a high concentration in the sensillum lymph surrounding olfactory neurons, are proposed to be important in pheromone detection and discrimination in insects. Here, we present a systematic study of PBP-ligand interaction kinetics. We find that PBP2, from the gypsy moth, Lymantria dispar, associates and dissociates slowly with its biofunctional ligands, (+)- and (-)-disparlure. Tryptophan anisotropy measurements detect PBP multimers in solution as well as an increase in the multimeric state of the protein upon long exposure to ligand. We propose a kinetic model that includes monomer/multimer equilibria and a two-step binding process: (1) external binding of the pheromone assisted by the C terminus of PBP2, and (2) slow embedding of the pheromone into the internal pocket. This experimentally derived model sheds light on the potential biological function and mechanism of PBPs as ligand scavengers.

  14. A quantitative kinetic model for the fast and isothermal hydrothermal liquefaction of Nannochloropsis sp.

    PubMed

    Hietala, David C; Faeth, Julia L; Savage, Phillip E

    2016-08-01

    Hydrothermal liquefaction (HTL) is a technology for converting algal biomass into biocrude oil and high-value products. To elucidate the underlying kinetics for this process, we conducted isothermal and non-isothermal reactions over a broad range of holding times (10s-60min), temperatures (100-400°C), and average heating rates (110-350°Cmin(-1)). Biocrude reached high yields (⩾37wt%) within 2min for heat-source set-point temperatures of 350°C or higher. We developed a microalgal HTL kinetic model valid from 10s to 60min, including significantly shorter timescales (10s-10min) than any previous model. The model predicts that up to 46wt% biocrude yields are achievable at 400°C and 1min, reaffirming the utility of short holding times and "fast" HTL. We highlight potential trade-offs between maximizing biocrude quantity and facilitating aqueous phase recovery, which may improve biocrude quality.

  15. Kinetic modeling of solid yields formation in the fast pyrolysis of mahogany wood

    NASA Astrophysics Data System (ADS)

    Wijayanti, W.; Sasongko, M. N.

    2016-03-01

    There have been many research of biomass pyrolysis not only in heat transfer point of view but also in chemical reaction point of view. In the present study, the rate of reaction (kinetic rate) formation of solid yield was calculated by varying the pyrolysis temperature that gives a chance of 250 °C, 350 °C, 450 °C, 500 °C, 600 °C, 700 °C, until 800°C with heating rate around 700 °C/hour. The heating rate used was the fast pyrolysis in which the heating rate for heating furnaces takes place quickly. Pyrolysis was accomplished by direct pyrolysis process in which each process was conducted at the certain pyrolysis temperature variation that took over 3 hours. Biomass used was mahogany wood, while the inert gas used to hold in order to avoid combustion was nitrogen gas. The decreasing of solid yields formation obtained was used to calculate the kinetic rate of the pyrolysis process. It was calculated by using the similar Arrhenius equation that considering the temperature changes during the process and the decreasing mass of solid yield formation occurred. The kinetic rate results showed the decomposition of biomass occurs tended in two stages, namely a stage of water evaporation and degradation of biomass solid yield coal followed by a stage of constant formation. The decomposition is expressed by the magnitude of the rate of reaction at 25˚C-517˚C temperature range with a reaction rate constant k1 = 2151.67 exp (-2141/Tp). While at pyrolysis temperatures above 517˚C, the reaction rate constant is expressed with k2 = 32.20 exp (-127.8 / Tp).

  16. Kinetics of fast short-term depression are matched to spike train statistics to reduce noise.

    PubMed

    Khanbabaie, Reza; Nesse, William H; Longtin, Andre; Maler, Leonard

    2010-06-01

    Short-term depression (STD) is observed at many synapses of the CNS and is important for diverse computations. We have discovered a form of fast STD (FSTD) in the synaptic responses of pyramidal cells evoked by stimulation of their electrosensory afferent fibers (P-units). The dynamics of the FSTD are matched to the mean and variance of natural P-unit discharge. FSTD exhibits switch-like behavior in that it is immediately activated with stimulus intervals near the mean interspike interval (ISI) of P-units (approximately 5 ms) and recovers immediately after stimulation with the slightly longer intervals (>7.5 ms) that also occur during P-unit natural and evoked discharge patterns. Remarkably, the magnitude of evoked excitatory postsynaptic potentials appear to depend only on the duration of the previous ISI. Our theoretical analysis suggests that FSTD can serve as a mechanism for noise reduction. Because the kinetics of depression are as fast as the natural spike statistics, this role is distinct from previously ascribed functional roles of STD in gain modulation, synchrony detection or as a temporal filter.

  17. Ligand-rebinding kinetics of 2/2 hemoglobin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

    PubMed

    Russo, Roberta; Giordano, Daniela; di Prisco, Guido; Hui Bon Hoa, Gaston; Marden, Michael C; Verde, Cinzia; Kiger, Laurent

    2013-09-01

    Kinetic studies were performed on ligand rebinding to a cold-adapted globin of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO). This 2/2 hemoglobin displays a rapid spectroscopic phase that is independent of CO concentration, followed by the standard bimolecular recombination. While the geminate recombination usually occurs on a ns timescale, Ph-2/2HbO displays a component of about 1μs that accounts for half of the geminate phase at 8°C, indicative of a relatively slow internal ligand binding. The O2 binding kinetics were measured in competition with CO to allow a short-time exposure of the deoxy hemes to O2 before CO replacement. Indeed Ph-2/2HbO is readily oxidised in the presence of O2, probably due to a superoxide character of the FeO2 bond induced by of a hydrogen-bond donor amino-acid residue. Upon O2 release or iron oxidation a distal residue (probably Tyr) is able to reversibly bind to the heme and as such to compete for binding with an external ligand. The transient hexacoordinated ferrous His-Fe-Tyr conformation after O2 dissociation could initiate the electron transfer from the iron toward its final acceptor, molecular O2 under our conditions. The hexacoordination via the distal Tyr is only partial, indicating a weak interaction between Tyr and the heme under atmospheric pressure. Hydrostatic high pressure enhances the hexacoordination indicating a flexible globin that allows structural changes. The O2 binding affinity for Ph-2/2HbO, poorly affected by the competition with Tyr, is about 1Torr at 8°C, pH7.0, which is compatible for an in vivo O2 binding function; however, this globin is more likely involved in a redox reaction associating diatomic ligands and their derived oxidative species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

  18. Binding conformation and kinetics of two pheromone-binding proteins from the Gypsy moth Lymantria dispar with biological and nonbiological ligands.

    PubMed

    Gong, Yongmei; Tang, Hao; Bohne, Cornelia; Plettner, Erika

    2010-02-02

    Pheromone-binding proteins (PBPs) in insects can bind various substances and selectively deliver the message of a signal molecule to the downstream components of the olfactory system. This can be achieved either through a ligand-specific conformational change of the C-terminal peptide of the PBP or by selectively binding/releasing the ligand. PBP may also act as a scavenger to protect the sensory neurons from saturating at high ligand doses. We have compared two PBPs from the gypsy moth (PBP1 and PBP2) and their truncated forms (TPBPs), which lack the C-terminal peptide, in this study. Stopped-flow kinetics with N-phenyl-1-naphthylamine (NPN) have revealed a diffusion-controlled collisional step, between PBP and NPN, after which the NPN relocates into a hydrophobic environment. This work supports the hypothesis that binding between PBPs and ligands occurs stepwise. With the method of tryptophan fluorescence quenching, we have shown different local conformational changes around Trp 37, induced by different ligands, manifested in changes of both the steric and electronic environment around the residue. Importantly, we have noticed a significant difference in the changes induced by the biological ligand (the pheromone) and nonbiological ligands. Therefore, we hypothesize that PBP may serve a different function in each kinetic step, displaying a unique P.L conformation.

  19. Thermodynamics and Kinetics of Ligand Binding to Vitamin B-12a: A Laboratory Experiment

    ERIC Educational Resources Information Center

    Sweigart, D. A.

    1975-01-01

    Describes an open-ended experiment involving a bioinorganic system that provides the student a direct link between thermodynamics and kinetics. It utilizes a rapid reaction technique and requires one to clearly understand the relationship of experimental observable (absorbance) to a mechanism. (GS)

  20. Thermodynamics and Kinetics of Ligand Binding to Vitamin B-12a: A Laboratory Experiment

    ERIC Educational Resources Information Center

    Sweigart, D. A.

    1975-01-01

    Describes an open-ended experiment involving a bioinorganic system that provides the student a direct link between thermodynamics and kinetics. It utilizes a rapid reaction technique and requires one to clearly understand the relationship of experimental observable (absorbance) to a mechanism. (GS)

  1. A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands

    PubMed Central

    Swinney, David C; Beavis, Paul; Chuang, Kai-Ting; Zheng, Yue; Lee, Ina; Gee, Peter; Deval, Jerome; Rotstein, David M; Dioszegi, Marianna; Ravendran, Palani; Zhang, Jun; Sankuratri, Surya; Kondru, Rama; Vauquelin, Georges

    2014-01-01

    BACKGROUND AND PURPOSE The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH Using [3H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor–ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k−2, was 1.2 × 10−3 min−1. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc–receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand. PMID:24628038

  2. Fast Photochemical Oxidation of Proteins (FPOP) for Comparing Structures of Protein/Ligand Complexes: The Calmodulin-peptide Model System

    PubMed Central

    Zhang, Hao; Gau, Brian C.; Jones, Lisa M.; Vidavsky, Ilan; Gross, Michael L.

    2010-01-01

    Fast Photochemical Oxidation of Proteins (FPOP) is a mass-spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive •OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one half the common amino acids on this time scale. Owing to the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex should provide structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows from a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not,. PMID:21142124

  3. Effects of various competing ligands on the kinetics of trace metal complexes of Laurentian Fulvic Acid in model solutions and natural waters.

    PubMed

    Yapici, Tahir; Fasfous, Ismail I; Zhao, Jiujiang; Chakrabarti, Chuni L

    2009-03-16

    The objective of this work was to study the effects of the following Ligands: Chelex-100, Dowex MAC-3 and Dowex 50WX-8 using Competing Ligand Exchange Method. This objective was achieved by investigating complex dissociation kinetics of trace metals: Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Mn(II) and Pb(II) of a well-characterized Laurentian Fulvic Acid (LFA) in model solutions and in a natural waters of Lake Heva (Québec, Canada). The effects of variation in the competing ligands (including their quantities) on the complex dissociation kinetics were quantitatively characterized by their first-order dissociation rate coefficients. The kinetic lability of the metal complexes varied with the metal-to-LFA ratio, as expected from the theory of metal complexes of the chemically and physically heterogeneous complexants, LFA. The general trend in the metal-binding by the above competing ligands was: Dowex 50WX-8>Chelex-100>Dowex MAC-3. However, no difference was found between the Dowex 50WX-8 and Chelex-100 for Cd(II), Zn(II), and Co(II). The results revealed the importance of the quantity of Chelex-100 as a competing ligand in the metal(II)-LFA complexation, on the dissociation kinetics of these complexes in model solutions. By developing Competing Ligand Exchange Method as an analytical technique, for studying the relative affinities of the above competing ligands for metals complexation in natural waters this work has made a substantial contribution to analytical chemistry.

  4. Kinetic theory for neuronal networks with fast and slow excitatory conductances driven by the same spike train.

    PubMed

    Rangan, Aaditya V; Kovacic, Gregor; Cai, David

    2008-04-01

    We present a kinetic theory for all-to-all coupled networks of identical, linear, integrate-and-fire, excitatory point neurons in which a fast and a slow excitatory conductance are driven by the same spike train in the presence of synaptic failure. The maximal-entropy principle guides us in deriving a set of three (1+1) -dimensional kinetic moment equations from a Boltzmann-like equation describing the evolution of the one-neuron probability density function. We explain the emergence of correlation terms in the kinetic moment and Boltzmann-like equations as a consequence of simultaneous activation of both the fast and slow excitatory conductances and furnish numerical evidence for their importance in correctly describing the coarse-grained dynamics of the underlying neuronal network.

  5. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics

    PubMed Central

    May, Lauren T.; Bridge, Lloyd J.; Stoddart, Leigh A.; Briddon, Stephen J.; Hill, Stephen J.

    2011-01-01

    A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A1 or A3 adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A3-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A1 and A3 receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min−1, respectively. At the A3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5′-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A3-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A1 receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC50 values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A3-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.—May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J., Hill, S. J. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics. PMID:21715680

  6. Nanohole-based SPR Instruments with Improved Spectral Resolution Quantify a Broad Range of Antibody-Ligand Binding Kinetics

    PubMed Central

    Im, Hyungsoon; Sutherland, Jamie N.; Maynard, Jennifer A.; Oh, Sang-Hyun

    2012-01-01

    We demonstrate an affordable low-noise SPR instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10−6 without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 milliseconds followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools. PMID:22235895

  7. Kinetics of proton diffusion in the regimes of fast and slow exchange between the membrane surface and the bulk solution.

    PubMed

    Medvedev, E S; Stuchebrukhov, A A

    2006-02-01

    The phenomenological model developed in our recent publications [9,10] is used to investigate the kinetics of proton diffusion from a source to a detector on the membrane surface. In most cases the observed kinetics shows a single diffusional maximum with the exponential ascending front and the power-law descending tail. The kinetics depends on the distance between the source and the detector. If the detector is located inside the proton collecting antenna, the kinetics corresponds to the surface diffusion at the times near the maximum and shortly thereafter, and it turns into the bulk diffusion kinetics at longer times, after the equilibrium is established between the membrane surface and the bulk solution. If the detector is located outside the antenna, the kinetics corresponds to the bulk diffusion at all times where the signal is nonvanishing. What is seen at locations near the antenna radius depends on the exchange regime. In the regime of fast exchange between the surface and the bulk as compared to the bulk diffusion, the kinetics shows a single peak whose location is intermediate between the peaks for the surface and bulk diffusion. In the regime of slow exchange there are two maxima corresponding to the surface and bulk diffusion. In buffered solutions the antenna radius decreases with increasing buffer concentration, which changes the kinetics from the surface to bulk diffusion. The theory is applied to interprete recent experiments on a phospholipid membrane [25]. It is found that (i) the fast exchange regime is operating since only a single maximum is observed; (ii) the shift of the maximum toward longer times with increasing buffer concentration is a manifestation of the transition from the surface to bulk diffusion kinetics.

  8. HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

    PubMed Central

    Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

    2010-01-01

    Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

  9. Stopped-flow study of anesthetic effect on water-transport kinetics through phospholipid membranes. Interfacial versus lipid core ligands.

    PubMed

    Inoue, T; Kamaya, H; Ueda, I

    1985-01-25

    We have compared ligand effects between polar and apolar anesthetic molecules upon water transport across phospholipid membranes by kinetic analysis of the osmotic swelling rate, using a stopped-flow technique. Chloroform and 1-hexanol were used as interfacial ligands, and carbon tetrachloride and n-hexane were used as their counterparts, representing lipid core action. Because anesthetics transform the solid-gel membrane into a liquid-crystalline state, and because phospholipid membranes display an anomaly in permeability at the phase transition, dimyristoylphosphatidylcholine vesicles were studied at temperatures above the main phase transition to avoid this anomaly. All these molecules increased the osmotic swelling rate. However, a significant difference was observed in the activation energy, delta Ep, between polar and apolar molecules; delta Ep was almost unaltered by the addition of polar molecules (chloroform and 1-hexanol), whereas it was decreased by apolar molecules (carbon tetrachloride and n-hexane). The obtained results were analyzed in terms of the dissolution-diffusion mechanism for water permeation across the lipid membrane. It is suggested that polar molecules affect water permeability by altering the partition of water between the membrane interior and water phase, and apolar molecules affect it by altering both the partition and the diffusion of water within the membrane interior.

  10. Migration kinetics of primary aromatic amines from polyamide kitchenware: Easy and fast screening procedure using fluorescence.

    PubMed

    Sanllorente, S; Sarabia, L A; Ortiz, M C

    2016-11-01

    Primary aromatic amines, PAAs, and their derivatives constitute a health risk and control of their migration from food contact materials is the subject of permanent attention by the authorities. 25.1% of notifications made by Rapid Alert System for Food and Feed in the European Union between 2010 and 2015 concerned PAAs, polyamide cooking utensils being a common source. It is thus useful to have fast and efficient analytical methods for their control. In this work a non-separative, easy, fast and inexpensive spectrofluorimetric method based on the second order calibration of excitation-emission fluorescence matrices (EEMs) was proposed for the determination of aniline (ANL), 2,4-diaminotoluene (2,4-TDA) and 4,4'-methylenedianiline (4,4'-MDA) in polyamide cooking utensils. The procedure made it possible to identify unequivocally each analyte. Trilinearity of the data tensor guarantees the uniqueness of the solution obtained through parallel factor analysis (PARAFAC), so the factors of the decomposition match up with the analytes. The three analytes were unequivocally identified by the correlation between the pure spectra and the PARAFAC excitation and emission spectral loadings. The recovery percentages found were, 82.6%, 112.7% and 84.4% for ANL, 2,4-TDA and 4,4'-MDA respectively. The proposed method was applied to carry out a migration test from polyamide cooking utensils, using a 3% (w/v) acetic acid in aqueous solution as food simulant. Detectable levels of 4,4'-MDA were found in food simulant from some of the investigated cooking utensils. Finally, a kinetic model for the migration of 4,4'-MDA has been fitted to experimental data obtained in the migration test. Thanks to the selectivity of PARAFAC calibration, which greatly simplifies sample treatment avoiding the use of toxic solvents, the developed method follows most green analytical chemistry principles.

  11. Three-dimensional lumbar segment kinetics of fast bowling in cricket.

    PubMed

    Ferdinands, René E D; Kersting, Uwe; Marshall, R N

    2009-08-07

    Cricket fast bowlers have a high incidence of serious lumbar injuries, such as lesions in the pars interarticularis. Kinematic studies have shown that bowling actions with large shoulder counter-rotation are associated with significantly higher incidences of lumbar injury. However, in bowling, there has been no calculation of the spinal loads, which are the causal mechanisms of such injuries. In this study, 21 fast bowlers (22.4+/-3.9 years) of premier grade level and above were tested using a three-dimensional (3D) motion analysis system. The mean ball release speed was 31.9+/-2.8 m s(-1) and ranged from 27.0 to 35.6 m s(-1). Kinematics and kinetics were calculated for lumbar spine lateral bending, rotation, and flexion during the delivery and power phases of bowling. Power calculations were used to define the actuation of lumbar spine motion as either active or controlled. The actuation of the lumbar spine was complex, involving multiple sequences of active and controlled motion. In addition, lumbar spine loads were largest during the power phase when the ground reaction forces were highest. In conclusion, the dynamic loads and the cyclical nature of their application when the spine is positioned near its end range of motion may be significant factors of injury to this region. In addition, the lumbar spine in bowling has to vigorously flex, laterally bend and rotate simultaneously in a complex interdependent sequence of actuation patterns. Therefore, any technical change to reduce injury susceptibility needs to consider the mechanics of whole body coordination and timing.

  12. Basic residue at position 14 is not required for fast assembly and disassembly kinetics in neural cadherin.

    PubMed

    Vunnam, Nagamani; Hammer, Nathan I; Pedigo, Susan

    2015-01-27

    In spite of their structural similarities, epithelial (E-) and neural (N-) cadherin are expressed at different types of synapses and differ significantly in their dimerization kinetics. Recent studies proposed a transient intermediate in E-cadherin as the key requirement for rapid disassembly kinetics of the adhesive dimer. This E-cadherin intermediate comprises four intermolecular ionic and H-bonding interactions between adhesive partners. These interactions are not preserved in N-cadherin except for a basic residue at the 14th position, which could stabilize the intermediate through either H-bonding or ionic interactions with the partner protomer. To investigate the origin of the rapid dimerization kinetics of N-cadherin in the presence of calcium, studies reported here systematically test the role of ionic and H-bonding interactions in dimerization kinetics using R14S, R14A, and R14E mutants of N-cadherin. Analytical size-exclusion chromatographic and bead aggregation studies showed two primary results. First, N-cadherin/R14S and N-cadherin/R14A mutants showed fast assembly and disassembly kinetics in the calcium-saturated state similar to that of wild-type N-cadherin. These results indicate that the fast disassembly of the calcium-saturated dimer of N-cadherin does not require a basic residue at the 14th position. Second, the dimerization kinetics of N-cadherin/R14E were slow in the calcium-saturated state, indicating that negative charge destabilizes the intermediate state. Taken together, these results indicate that the basic residue at the 14th position does not promote rapid dimerization kinetics but that an acidic amino acid in that position significantly impairs dimerization kinetics.

  13. Validity of model approximations for receptor-ligand kinetics in nuclear medicine

    SciTech Connect

    Salinas, Cristian A.; Muzic, Raymond F. Jr.; Saidel, Gerald M.

    2007-05-15

    An appropriate mathematical model is required for quantitative analysis of high affinity radioligands as direct or surrogate probes to measure receptor distribution, affinity, concentration, binding potential, and endogenous or exogenous ligand occupancy levels. For studies with positron emission tomography (PET) or single photon emission computed tomography (SPECT), the receptor-ligand compartment model has been well established and widely used. This pharmacokinetic model is represented mathematically by a set of nonlinear ordinary differential equations. Variations of models for PET and SPECT account for radioactive decay differently. These are not equivalent and entail assumptions or approximations that may be not appreciated. In this study, a general form of the model is presented and compared with others with various approximations, which are valid only under specific conditions. The various approximate formulations were analytically compared to the exact model to identify the terms that were neglected in the approximate formulations. The extent to which the approximations impact the model solutions was assessed by computer simulations based on numerical solutions to each set of equations. Specifically, each model formulation was tested using three simulated injection protocols representing a typical PET experiment, a typical SPECT experiment, and an extreme experiment where both the injected activity and the specific activity were very high. No significant differences were found among the output of the three model formulations when the PET and SPECT injection protocols were tested. The only conditions that produced significant differences occurred when the specific activity and the administered activity were simultaneously very high. These conditions, however, have little practical relevance to experimentally achievable conditions due to radiation dose and specific activity of radiopharmaceuticals.

  14. Predicting properties of gas and solid streams by intrinsic kinetics of fast pyrolysis of wood

    SciTech Connect

    Klinger, Jordan; Bar-Ziv, Ezra; Shonnard, David; Westover, Tyler; Emerson, Rachel

    2015-12-12

    Pyrolysis has the potential to create a biocrude oil from biomass sources that can be used as fuel or as feedstock for subsequent upgrading to hydrocarbon fuels or other chemicals. The product distribution/composition, however, is linked to the biomass source. This work investigates the products formed from pyrolysis of woody biomass with a previously developed chemical kinetics model. Different woody feedstocks reported in prior literature are placed on a common basis (moisture, ash, fixed carbon free) and normalized by initial elemental composition through ultimate analysis. Observed product distributions over the full devolatilization range are explored, reconstructed by the model, and verified with independent experimental data collected with a microwave-assisted pyrolysis system. These trends include production of permanent gas (CO, CO2), char, and condensable (oil, water) species. Elementary compositions of these streams are also investigated. As a result, close agreement between literature data, model predictions, and independent experimental data indicate that the proposed model/method is able to predict the ideal distribution from fast pyrolysis given reaction temperature, residence time, and feedstock composition.

  15. Fast kinetics of magnesium monochloride cations in interlayer-expanded titanium disulfide for magnesium rechargeable batteries

    DOE PAGES

    Yoo, Hyun Deog; Liang, Yanliang; Dong, Hui; ...

    2017-08-24

    Magnesium rechargeable batteries potentially offer high-energy density, safety, and low cost due to the ability to employ divalent, dendrite-free, and earth-abundant magnesium metal anode. Despite recent progress, further development remains stagnated mainly due to the sluggish scission of magnesium-chloride bond and slow diffusion of divalent magnesium cations in cathodes. Here in this paper we report a battery chemistry that utilizes magnesium monochloride cations in expanded titanium disulfide. Combined theoretical modeling, spectroscopic analysis, and electrochemical study reveal fast diffusion kinetics of magnesium monochloride cations without scission of magnesium-chloride bond. The battery demonstrates the reversible intercalation of 1 and 1.7 magnesium monochloridemore » cations per titanium at 25 and 60 °C, respectively, corresponding to up to 400 mAh g-1 capacity based on the mass of titanium disulfide. The large capacity accompanies with excellent rate and cycling performances even at room temperature, opening up possibilities for a variety of effective intercalation hosts for multivalent-ion batteries.« less

  16. Predicting properties of gas and solid streams by intrinsic kinetics of fast pyrolysis of wood

    DOE PAGES

    Klinger, Jordan; Bar-Ziv, Ezra; Shonnard, David; ...

    2015-12-12

    Pyrolysis has the potential to create a biocrude oil from biomass sources that can be used as fuel or as feedstock for subsequent upgrading to hydrocarbon fuels or other chemicals. The product distribution/composition, however, is linked to the biomass source. This work investigates the products formed from pyrolysis of woody biomass with a previously developed chemical kinetics model. Different woody feedstocks reported in prior literature are placed on a common basis (moisture, ash, fixed carbon free) and normalized by initial elemental composition through ultimate analysis. Observed product distributions over the full devolatilization range are explored, reconstructed by the model, andmore » verified with independent experimental data collected with a microwave-assisted pyrolysis system. These trends include production of permanent gas (CO, CO2), char, and condensable (oil, water) species. Elementary compositions of these streams are also investigated. As a result, close agreement between literature data, model predictions, and independent experimental data indicate that the proposed model/method is able to predict the ideal distribution from fast pyrolysis given reaction temperature, residence time, and feedstock composition.« less

  17. Kinetics of thermolysis of some transition metal nitrate complexes with 1,6-diaminohexane ligand.

    PubMed

    Singh, Gurdip; Singh, Chandra Prakash; Mannan, S M

    2006-07-31

    Metal nitrate complexes of general formula [M(dah)(2)](NO(3))(2) (where M=Zn, Cu and Ni; dah=1,6-diaminohexane) have been prepared and characterized by elemental analysis, infrared spectroscopy (IR) and gravimetric method. The thermal decomposition has been studied using thermogravimetry (TG). Simultaneous thermogravimetry-differential thermogravimetry-differential thermal analysis (TG-DTG-DTA) and differential scanning calorimetry (DSC) were done in N(2) atmosphere. Isothermal TG of initial decomposition of all these complexes, have been carried out to evaluate the kinetics of early thermolysis. Both, model fitting and isoconversional method have been used for the evaluation of the kinetics of thermal decomposition. Model fitting method have given the single value of activation energy (E) whereas, isoconversional method yields a series of E-value, which vary with extent of conversion. Ignition of the complexes was measured to see the response towards rapid heating with varying amounts. The thermal stability of the complexes was found to be in the order as [Zn(dah)(2)](NO(3))(2)>[Cu(dah)(2)](NO(3))(2) approximately [Ni(dah)(2)](NO(3))(2).

  18. Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

    PubMed

    Xia, Lizi; de Vries, Henk; IJzerman, Ad P; Heitman, Laura H

    2016-03-01

    Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.

  19. Kinetics of brucite dissolution at 25°C in the presence of organic and inorganic ligands and divalent metals

    NASA Astrophysics Data System (ADS)

    Pokrovsky, Oleg S.; Schott, Jacques; Castillo, Alain

    2005-02-01

    Brucite (Mg(OH) 2) dissolution rate was measured at 25°C in a mixed-flow reactor at various pH (5 to 11) and ionic strengths (0.01 to 0.03 M) as a function of the concentration of 15 organic and 5 inorganic ligands and 8 divalent metals. At neutral and weakly alkaline pH, the dissolution is promoted by the addition of the following ligands ranked by decreasing effectiveness: EDTA ≥ H 2PO 4- > catechol ≥ HCO 3- > ascorbate > citrate > oxalate > acetate ˜ lactate and it is inhibited by boric acid. At pH >10.5, it decreases in the presence of PO 43-, CO 32-, F -, oxine, salicylate, lactate, acetate, 4-hydroxybenzoate, SO 42- and B(OH) 4- with orthophosphate and borate being the strongest and the weakest inhibitor, respectively. Xylose (up to 0.1 M), glycine (up to 0.05 M), formate (up to 0.3 M) and fulvic and humic acids (up to 40 mg/L DOC) have no effect on brucite dissolution kinetics. Fluorine inhibits dissolution both in neutral and alkaline solutions. From F sorption experiments in batch and flow-through reactors and the analysis of reacted surfaces using X-ray Photoelectron Spectroscopy (XPS), it is shown that fluorine adsorption is followed by its incorporation in brucite lattice likely via isomorphic substitution with OH. The effect of eight divalent metals (Sr, Ba, Ca, Pb, Mn, Fe, Co and Ni) studied at pH 4.9 and 0.01 M concentration revealed brucite dissolution rates to be correlated with the water molecule exchange rates in the first hydration sphere of the corresponding cation. The effect of investigated ligands on brucite dissolution rate can be modelled within the framework of the surface coordination approach taking into account the adsorption of ligands on dissolution-active sites and the molecular structure of the surface complexes they form. The higher the value of the ligand sorption constant, the stronger will be its catalyzing or inhibiting effect. As for Fe and Al oxides, bi- or multidentate mononuclear surface complexes, that labilize Mg

  20. Probability description of ligand-receptor interactions. Evaluation of reliability of events with small and supersmall doses. I. Kinetics of ligand-receptor interactions.

    PubMed

    Gurevich, K G; Varfolomeev, S D

    1999-09-01

    We have developed mathematical methods for describing ligand-receptor interactions (LRI) using Markov chains. Under some conditions, the mean value of ligand-receptor complexes obtained using Markov chains coincides with that obtained from the law of mass action. Using the calculated ratio of standard deviation to mean number of ligand-receptor complexes, we show that with small concentrations of ligand-receptor complexes LRI must be described using probability methods. Using data from the literature, we show that LRI description using the mass-action law under these conditions can cause significant errors in interpretation of experimental data.

  1. A Pyridine-Based Ligand with Two Hydrazine Functions for Lanthanide Chelation: Remarkable Kinetic Inertness for a Linear, Bishydrated Complex.

    PubMed

    Bonnet, Célia S; Laine, Sophie; Buron, Frédéric; Tircsó, Gyula; Pallier, Agnès; Helm, Lothar; Suzenet, Franck; Tóth, Éva

    2015-06-15

    To study the influence of hydrazine functions in the ligand skeleton, we designed the heptadentate HYD ligand (2,2',2″,2‴-(2,2'-(pyridine-2,6-diyl)bis(2-methylhydrazine-2,1,1-triyl)) tetraacetic acid) and compared the thermodynamic, kinetic, and relaxation properties of its Ln(3+) complexes to those of the parent pyridine (Py) analogues without hydrazine (Py = 2,6-pyridinebis(methanamine)-N,N,N',N'-tetraacetic acid). The protonation constants of HYD were determined by pH-potentiometric measurements, and assigned by a combination of UV-visible and NMR spectroscopies. The protonation sequence is rather unusual and illustrates that small structural changes can strongly influence ligand basicity. The first protonation step occurs on the pyridine nitrogen in the basic region, followed by two hydrazine nitrogens and the carboxylate groups at acidic pH. Contrary to Py, HYD self-aggregates through a pH-dependent process (from pH ca. 4). Thermodynamic stability constants have been obtained by pH-potentiometry and UV-visible spectrophotometry for various Ln(3+) and physiological cations (Zn(2+), Ca(2+), Cu(2+)). LnHYD stability constants show the same trend as those of LnDTPA complexes along the Ln(3+) series, with log K = 18.33 for Gd(3+), comparable to the Py analogue. CuHYD has a particularly high stability (log K > 19) preventing its determination from pH-potentiometric measurements. The stability constant of CuPy was also revisited and found to be underestimated in previous studies, highlighting that UV-visible spectrophotometry is often indispensable to obtain reliable stability constants for Cu(2+) chelates. The dissociation of GdL, assessed by studying the Cu(2+)-exchange reaction, occurs mainly via an acid-catalyzed process, with limited contribution from direct Cu(2+) attack. The kinetic inertness of GdHYD is remarkable for a linear bishydrated chelate; the 25-fold increase in the dissociation half-life with respect to the monohydrated commercial contrast agent

  2. Gyrokinetic electron and fully kinetic ion simulations of fast magnetosonic waves in the magnetosphere

    NASA Astrophysics Data System (ADS)

    Gao, Xiaotian; Liu, Kaijun; Wang, Xueyi; Min, Kyungguk; Lin, Yu; Wang, Xiaogang

    2017-06-01

    Two-dimensional simulations using a gyrokinetic electron and fully kinetic ion (GeFi) scheme are preformed to study the excitation of fast magnetosonic waves in the terrestrial magnetosphere, which arise from the ion Bernstein instability driven by proton velocity distributions with a positive slope with respect to the perpendicular velocity. Since both ion and electron kinetics are relevant, particle-in-cell (PIC) simulations have often been employed to study the wave excitation. However, the full particle-in-cell scheme is computationally expensive for simulating waves in the ion scale because the electron scale must be fully resolved. Therefore, such simulations are limited to reduced proton-to-electron mass ratio ( m p / m e) and light-to-Alfvén speed ratio ( c / v A). The present study exploits the GeFi scheme that can break through these limitations to some extent, so larger m p / m e and c / v A can be used. In the simulations presented, the ion Bernstein instability is driven by a proton velocity distribution composed of 10% energetic protons with a shell distribution and 90% relatively cool, background protons with a Maxwellian distribution. The capability of the GeFi code in simulating the ion Bernstein instability is first demonstrated by comparing a GeFi simulation using reduced mass ratio ( m p / m e = 100) and speed ratio ( c / v A = 15) to a corresponding PIC simulation as well as linear dispersion analysis. A realistic speed ratio ( c / v A = 400) and a larger mass ratio ( m p / m e = 400) are then adopted in the GeFi code to explore how the results vary. It is shown that, as the increased m p / m e and c / v A lead to a larger lower hybrid frequency, ion Bernstein waves are excited at more ion cyclotron harmonics, consistent with the general prediction of linear dispersion theory. On the other hand, the GeFi simulations also revealed some interesting features after the instability saturation, which are likely related to nonlinear wave

  3. Water-soluble Mo3S4 clusters bearing hydroxypropyl diphosphine ligands: synthesis, crystal structure, aqueous speciation, and kinetics of substitution reactions.

    PubMed

    Basallote, Manuel G; Fernández-Trujillo, M Jesús; Pino-Chamorro, Jose Ángel; Beltrán, Tomás F; Corao, Carolina; Llusar, Rosa; Sokolov, Maxim; Vicent, Cristian

    2012-06-18

    The [Mo(3)S(4)Cl(3)(dhprpe)(3)](+) (1(+)) cluster cation has been prepared by reaction between Mo(3)S(4)Cl(4)(PPh(3))(3) (solvent)(2) and the water-soluble 1,2-bis(bis(hydroxypropyl)phosphino)ethane (dhprpe, L) ligand. The crystal structure of [1](2)[Mo(6)Cl(14)] has been determined by X-ray diffraction methods and shows the typical incomplete cuboidal structure with a capping and three bridging sulfides. The octahedral coordination around each metal center is completed with a chlorine and two phosphorus atoms of the diphosphine ligand. Depending on the pH, the hydroxo group of the functionalized diphosphine can substitute the chloride ligands and coordinate to the cluster core to give new clusters with tridentate deprotonated dhprpe ligands of formula [Mo(3)S(4)(dhprpe-H)(3)](+) (2(+)). A detailed study based on stopped-flow, (31)P{(1)H} NMR, and electrospray ionization mass spectrometry techniques has been carried out to understand the behavior of acid-base equilibria and the kinetics of interconversion between the 1(+) and the 2(+) forms. Both conversion of 1(+) to 2(+) and its reverse process occur in a single kinetic step, so that reactions proceed at the three metal centers with statistically controlled kinetics. The values of the rate constants under different conditions are used to discuss on the mechanisms of opening and closing of the chelate rings with coordination or dissociation of chloride.

  4. Kinetic and Spectral Properties of Isovaleryl-CoA Dehydrogenase and Interaction with Ligands

    PubMed Central

    Mohsen, Al-Walid A.; Vockley, Jerry

    2014-01-01

    Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF•min−1•mg−1, which was ~20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 µM and 4.3 × 106•M−1•sec−1 per monomer, respectively, and its Km for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KD app) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropylacetyl-CoA, and ETF induced CD spectral changes at the 250–500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM. PMID:25450250

  5. Carbohydrazones as new class of carbonic anhydrase inhibitors: Synthesis, kinetics, and ligand docking studies.

    PubMed

    Iqbal, Sarosh; Saleem, Muhammad; Azim, M Kamran; Taha, Muhammad; Salar, Uzma; Khan, Khalid Mohammed; Perveen, Shahnaz; Choudhary, M Iqbal

    2017-06-01

    Discovery and development of carbonic anhydrase inhibitors is crucial for their clinical use as antiepileptic, diurectic and antiglaucoma agents. Keeping this in mind, we have synthesized carbohydrazones 1-27 and evaluated them for their in vitro carbonic anhydrase inhibitory potential. Out of twenty-seven compounds, compounds 1 (IC50=1.33±0.01µM), 2 (IC50=1.85±0.24µM), 3 (IC50=1.37±0.06µM), and 9 (IC50=1.46±0.12µM) have showed carbonic anhydrase inhibition better than the standard drug zonisamide (IC50=1.86±0.03µM). Moreover, compounds 4 (IC50=2.32±0.04µM), 5 (IC50=3.96±0.35µM), 7 (IC50=2.33±0.02µM), and 8 (IC50=2.67±0.01µM) showed good inhibitory activity. Cheminformatic analysis has shown that compounds 1 and 2 possess lead-like properties. In addition, kinetic and molecular docking studies were also performed to investigate the binding interaction between carbohydrazones and carbonic anhydrase enzyme. This study has identified a novel and potent class of carbonic anhydrase inhibitors with the potential to be investigated further. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Kinetic, dynamic, ligand binding properties, and structural models of a dual-substrate specific nudix hydrolase from Schizosaccharomyces pombe.

    PubMed

    Garza, John A; Ilangovan, Udayar; Hinck, Andrew P; Barnes, Larry D

    2009-07-07

    Schizosaccharomyces pombe Aps1 is a nudix hydrolase that catalyzes the hydrolysis of both diadenosine 5',5'''-P(1),P(n)-oligophosphates and diphosphoinositol polyphosphates in vitro. Nudix hydrolases act upon a wide variety of substrates, despite having a common 23 amino acid catalytic motif; hence, the residues responsible for substrate specificity are considered to reside outside the common catalytic nudix motif. The specific residues involved in binding each substrate of S. pombe Aps1 are unknown. In this study, we have conducted mutational and kinetic studies in combination with structural homology modeling and NMR spectroscopic analyses to identify potential residues involved in binding each class of substrates. This study demonstrates several major findings with regard to Aps1. First, the determination of the kinetic parameters of K(m) and k(cat) indicated that the initial 31 residues of Aps1 are not involved in substrate binding or catalysis with respect to Ap(6)A. Second, NMR spectroscopic analyses revealed the secondary structure and three dynamic backbone regions, one of which corresponds to a large insert in Aps1 as compared to other putative fungal orthologues. Third, two structural models of Aps1Delta2-19, based on the crystal structures of human DIPP1 and T. thermophilus Ndx1, were generated using homology modeling. The structural models were in excellent agreement with the NMR-derived secondary structure of Aps1Delta2-19. Fourth, NMR chemical shift mapping in conjunction with structural homology models indicated several residues outside the catalytic nudix motif that are involved in specific binding of diphosphoinositol polyphosphate or diadenosine oligophosphate ligands.

  7. Ectopic Expression of Mouse Melanopsin in Drosophila Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation.

    PubMed

    Yasin, Bushra; Kohn, Elkana; Peters, Maximilian; Zaguri, Rachel; Weiss, Shirley; Schopf, Krystina; Katz, Ben; Huber, Armin; Minke, Baruch

    2017-03-03

    The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was ∼40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Ca2+_, Sr2+_force relationships and kinetic properties of fast-twitch rat leg muscle fibre subtypes.

    PubMed

    Galler, S

    1999-10-01

    Force generation of fast-twitch and slow-twitch fibres exhibits large differences in its sensitivity to Ca2+ and Sr2+ (e.g. Fink et al. 1986). Little is known about fast-twitch fibre subtypes. Thus, a variety of mechanical measurements on segments of rehydrated freeze-dried fast-twitch rat leg muscle fibres were executed in this study. Among these, the Ca2+- and Sr2+-force relationship and the unloaded shortening velocity were determined. The fibres were classified into subtypes according to their kinetics of stretch activation (Galler et al. 1994). In all fibres, the maximal force under Sr2+ activation was about 0.9 of that under Ca2+ activation. The Ca2+- and Sr2+-force relationship exhibited a biphasic shape with a steeper part (Hill coefficient, n1) below 50% and a flatter part (Hill coefficient, n2) above 50% of maximal force. The difference between the Ca2+ - and Sr2+ -sensitivity was independent of the fibre subtypes. The Hill coefficients were only partially correlated with kinetic properties. The correlation was more pronounced for the unloaded shortening velocity than for the kinetics of stretch activation. The data are consistent with the idea that the Ca2+ and Sr2+ sensitivities of fast-twitch fibres are mainly determined by a single isoform of troponin C. Among several protein isoforms, the isoforms of the myosin light chains seem to be involved for determining the slope of the Ca2+- and Sr2+-force relationship of fast-twitch muscle fibres.

  9. 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…

  10. 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…

  11. 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

  12. One-dimensional biomass fast pyrolysis model with reaction kinetics integrated in an Aspen Plus Biorefinery Process Model

    DOE PAGES

    Humbird, David; Trendewicz, Anna; Braun, Robert; ...

    2017-01-12

    A biomass fast pyrolysis reactor model with detailed reaction kinetics and one-dimensional fluid dynamics was implemented in an equation-oriented modeling environment (Aspen Custom Modeler). Portions of this work were detailed in previous publications; further modifications have been made here to improve stability and reduce execution time of the model to make it compatible for use in large process flowsheets. The detailed reactor model was integrated into a larger process simulation in Aspen Plus and was stable for different feedstocks over a range of reactor temperatures. Sample results are presented that indicate general agreement with experimental results, but with higher gasmore » losses caused by stripping of the bio-oil by the fluidizing gas in the simulated absorber/condenser. Lastly, this integrated modeling approach can be extended to other well-defined, predictive reactor models for fast pyrolysis, catalytic fast pyrolysis, as well as other processes.« less

  13. Differences in mechano-transducer channel kinetics underlie tonotopic distribution of fast adaptation in auditory hair cells.

    PubMed

    Ricci, Anthony

    2002-04-01

    The first step in audition is a deflection of the sensory hair bundle that opens mechanically gated channels, depolarizing the sensory hair cells. Two mechanism of adaptation of mechano-electric transducer (MET) channels have been identified in turtle auditory hair cells. The rate of fast adaptation varies tonotopically and is postulated to underlie a mechanical tuning mechanism in turtle auditory hair cells. Fast adaptation is driven by a calcium-dependent feedback process associated with MET channels. The purpose of this paper is to test the hypothesis that fast adaptation contributes to MET channel kinetics and that variations in channel kinetics underlie the tonotopic distribution of fast adaptation. To test for kinetic differences, the open channel blocker dihydrostreptomycin (DHS) was used. DHS blocked MET currents from low-frequency cells (IC(50) = 14 +/- 2 microM) more effectively than high-frequency cells (IC(50) = 75 +/- 5 microM), suggesting differences in MET channel properties. DHS block showed similar calcium sensitivities at both papilla locations. No difference in calcium permeation or block of the transducer channels was observed, indicating that the DHS effect was not due to differences in the channel pore. Slowing adaptation increased DHS efficacy, and speeding adaptation decreased DHS efficacy, suggesting that adaptation was influencing DHS block. DHS block of MET channels slowed adaptation, most likely by reducing the peak intraciliary calcium concentration achieved, supporting the hypothesis that the rate of adaptation varies with the calcium load per stereocilia. Another channel blocker, amiloride showed similar efficacy for high- and low-frequency cells with an IC(50) of 24.2 +/- 0.5 microM and a Hill coefficient of 2 but appeared to block high-frequency channels faster than low-frequency channels. To further explore MET channel differences between papilla locations, stationary noise analysis was performed. Spectral analysis of the noise

  14. Kinetics of Ligand-Receptor Interaction Reveals an Induced-Fit Mode of Binding in a Cyclic Nucleotide-Activated Protein

    PubMed Central

    Peuker, Sebastian; Cukkemane, Abhishek; Held, Martin; Noé, Frank; Kaupp, U. Benjamin; Seifert, Reinhard

    2013-01-01

    Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼107 M−1 s−1 are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding. PMID:23332059

  15. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

    PubMed

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

    2016-09-02

    The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

  16. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

    PubMed Central

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

    2016-01-01

    ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

  17. Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway.

    PubMed

    Powers, L; Chance, B; Chance, M; Campbell, B; Friedman, J; Khalid, S; Kumar, C; Naqui, A; Reddy, K S; Zhou, Y

    1987-07-28

    Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.

  18. Kinetic-MHD hybrid simulation of fishbone modes excited by fast ions on the experimental advanced superconducting tokamak (EAST)

    NASA Astrophysics Data System (ADS)

    Pei, Youbin; Xiang, Nong; Hu, Youjun; Todo, Y.; Li, Guoqiang; Shen, Wei; Xu, Liqing

    2017-03-01

    Kinetic-MagnetoHydroDynamic hybrid simulations are carried out to investigate fishbone modes excited by fast ions on the Experimental Advanced Superconducting Tokamak. The simulations use realistic equilibrium reconstructed from experiment data with the constraint of the q = 1 surface location (q is the safety factor). Anisotropic slowing down distribution is used to model the distribution of the fast ions from neutral beam injection. The resonance condition is used to identify the interaction between the fishbone mode and the fast ions, which shows that the fishbone mode is simultaneously in resonance with the bounce motion of the trapped particles and the transit motion of the passing particles. Both the passing and trapped particles are important in destabilizing the fishbone mode. The simulations show that the mode frequency chirps down as the mode reaches the nonlinear stage, during which there is a substantial flattening of the perpendicular pressure of fast ions, compared with that of the parallel pressure. For passing particles, the resonance remains within the q = 1 surface, while, for trapped particles, the resonant location moves out radially during the nonlinear evolution. In addition, parameter scanning is performed to examine the dependence of the linear frequency and growth rate of fishbones on the pressure and injection velocity of fast ions.

  19. A survey of the cusp ion outflow's kinetic energy flux measured by Polar and FAST during conjunction events

    NASA Astrophysics Data System (ADS)

    Tian, S.; Wygant, J. R.; Cattell, C. A.; Scudder, J. D.; McFadden, J. P.; Mozer, F.; Russell, C. T.

    2014-12-01

    Polar and FAST conjunction events are selected from Polar cusp crossings in 1997. These conjunction events reveal a common pattern in which Polar observed significant ion kinetic energy flux in the upward direction at mid-altitudes (below 6 Re). Depending on the magnetic activity level, the maximum ion kinetic energy flux is on the order of 10-100 mW/m^2, when mapped to the ionosphere. It is an order of magnitude or more larger than the ion kinetic energy flux observed by FAST in conjunction at altitudes of <1 Re. Therefore, the ion outflows are significantly energized within the mid-latitude cusp. Also shown in the conjunction events is that the downward Poynting flux has enough wave energy to power the ion energization. The observed pattern suggests that the cusp at ionosphere altitudes is not a simple mapping of higher altitude particles. Instead, the mid-latitude cusp receives significant downward Poynting flux from higher altitude due to the solar wind/magnetosphere coupling. Within the mid-altitude cusp, the Poynting flux then supplies energy to power the ionosphere/magnetosphere coupling. Ion outflows are triggered and energized, forming a planetary wind that feeds the magnetosphere with ionospheric ions. During southward IMF, the wind convects anti-sunward and can affect the tail lobe, the nightside auroral region and the nightside plasma sheet.

  20. Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics.

    PubMed

    Baker, Bradley J; Jin, Lei; Han, Zhou; Cohen, Lawrence B; Popovic, Marko; Platisa, Jelena; Pieribone, Vincent

    2012-07-15

    A substantial increase in the speed of the optical response of genetically encoded fluorescent protein voltage sensors (FP voltage sensors) was achieved by using the voltage-sensing phosphatase genes of Nematostella vectensis and Danio rerio. A potential N. vectensis voltage-sensing phosphatase was identified in silico. The voltage-sensing domain (S1-S4) of the N. vectensis homolog was used to create an FP voltage sensor called Nema. By replacing the phosphatase with a cerulean/citrine FRET pair, a new FP voltage sensor was synthesized with fast off kinetics (Tau(off)<5ms). However, the signal was small (ΔF/F=0.4%/200mV). FP voltage sensors using the D. rerio voltage-sensing phosphatase homolog, designated Zahra and Zahra 2, exhibited fast on and off kinetics within 2ms of the time constants observed with the organic voltage-sensitive dye, di4-ANEPPS. Mutagenesis of the S4 region of the Danio FP voltage sensor shifted the voltage dependence to more negative potentials but did not noticeably affect the kinetics of the optical signal.

  1. Structural basis for a hand-like site in the calcium sensor CatchER with fast kinetics

    SciTech Connect

    Zhang, Ying; Reddish, Florence; Tang, Shen; Zhuo, You; Wang, Yuan-Fang; Yang, Jenny J.; Weber, Irene T.

    2013-12-01

    High-resolution crystal structures of the designed calcium sensor CatchER revealed snapshots of calcium and gadolinium ions binding within the designed site in agreement with its fast kinetics. Calcium ions, which are important signaling molecules, can be detected in the endoplasmic reticulum by an engineered mutant of green fluorescent protein (GFP) designated CatchER with a fast off-rate. High resolution (1.78–1.20 Å) crystal structures were analyzed for CatchER in the apo form and in complexes with calcium or gadolinium to probe the binding site for metal ions. While CatchER exhibits a 1:1 binding stoichiometry in solution, two positions were observed for each of the metal ions bound within the hand-like site formed by the carboxylate side chains of the mutated residues S147E, S202D, Q204E, F223E and T225E that may be responsible for its fast kinetic properties. Comparison of the structures of CatchER, wild-type GFP and enhanced GFP confirmed that different conformations of Thr203 and Glu222 are associated with the two forms of Tyr66 of the chromophore which are responsible for the absorbance wavelengths of the different proteins. Calcium binding to CatchER may shift the equilibrium for conformational population of the Glu222 side chain and lead to further changes in its optical properties.

  2. Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca(2+) -regulated Photoproteins of Different Organisms.

    PubMed

    Eremeeva, Elena V; Bartsev, Sergey I; van Berkel, Willem J H; Vysotski, Eugene S

    2017-03-01

    Upon binding their metal ion cofactors, Ca(2+) -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca(2+) -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca(2+) -regulated photoproteins.

  3. Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.

    PubMed

    Kobori, Shungo; Ichihashi, Norikazu; Kazuta, Yasuaki; Matsuura, Tomoaki; Yomo, Tetsuya

    2012-08-01

    Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two on-switch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.

  4. A comparison of the short-term kinetics of zinc metabolism in women during fasting and following a breakfast meal.

    PubMed

    Lowe, N M; Woodhouse, L R; King, J C

    1998-10-01

    The physiological importance and mechanism of the postprandial fall in plasma Zn concentration is not well understood. In order to gain further information on this apparent redistribution of plasma Zn, a stable isotope, 70Zn, was used to study the effect of a breakfast meal on plasma Zn kinetics. Nine women participated in two trials, a fasting trial and a breakfast-meal trial; five of the women participated in a third trial in which the energy content of the breakfast meal was doubled. At each trial, 0.1 mg of 70Zn was infused intravenously, and the plasma disappearance of the isotope was analysed using a two-compartment model of Zn kinetics. Plasma Zn concentration fell significantly following the two trials in which the subjects were given meals, reaching low points that were 13 and 19%, respectively, below concentrations at comparable times during the fasting trial. Kinetic analysis revealed that after the doubled breakfast meal there was a significant fall (P < 0.007) in the size of the most rapidly turning over Zn pool (pool (a)) from 2.90 (SE 0.13) mg in the fasting state to 2.47 (SE 0.14) mg postprandially. The fractional turnover rate of pool (a) to other extravascular Zn pools, i.e. outside the two-compartment system, was also significantly elevated after the doubled breakfast meal (P < 0.05). These results suggest that the decline in plasma Zn concentration following a meal is due to a redistribution of Zn from the plasma to other more slowly turning over extravascular pools that may be involved in the assimilation and metabolism of fuels following food intake.

  5. Integrated Kinetic Simulation of Laser-Plasma Interactions, Fast-Electron Generation and Transport in Fast Ignition

    SciTech Connect

    Kemp, A; Cohen, B; Divol, L

    2009-11-16

    We present new results on the physics of short-pulse laser-matter interaction of kilojoule-picosecond pulses at full spatial and temporal scale, using a new approach that combines a 3D collisional electromagnetic Particle-in-Cell code with an MHD-hybrid model of high-density plasma. In the latter, collisions damp out plasma waves, and an Ohm's law with electron inertia effects neglected determines the electric field. In addition to yielding orders of magnitude in speed-up while avoiding numerical instabilities, this allows us to model the whole problem in a single unified framework: the laser-plasma interaction at sub-critical densities, energy deposition at relativistic critical densities, and fast-electron transport in solid densities. Key questions such as the multi-picosecond temporal evolution of the laser energy conversion into hot electrons, the impact of return currents on the laser-plasma interaction, and the effect of self-generated electric and magnetic fields on electron transport will be addressed. We will report applications to current experiments.

  6. Integrated kinetic simulation of laser-plasma interactions, fast-electron generation, and transport in fast ignition

    SciTech Connect

    Kemp, A. J.; Cohen, B. I.; Divol, L.

    2010-05-15

    We present new results on the physics of short-pulse laser-matter interaction of kilojoule-picosecond pulses at full spatial and temporal scale using a new approach that combines a three-dimensional collisional electromagnetic particle-in-cell code with a magnetohydrodynamic-hybrid model of high-density plasma. In the latter, collisions damp out plasma waves, and an Ohm's law with electron inertia effects neglected determines the electric field. In addition to yielding orders of magnitude in speed-up while avoiding numerical instabilities, this allows us to model the whole problem in a single unified framework: the laser-plasma interaction at subcritical densities, energy deposition at relativistic critical densities, and fast- electron transport in solid densities. Key questions such as the multipicosecond temporal evolution of the laser energy conversion into hot electrons, the impact of return currents on the laser-plasma interaction, and the effect of self-generated electric and magnetic fields on electron transport will be addressed. We will report applications to current experiments.

  7. Kinetic identification of protein ligands in a 51,200 small-molecule library using microarrays and a label-free ellipsometric scanner

    NASA Astrophysics Data System (ADS)

    Landry, James P.; Proudian, Andrew P.; Malovichko, Galina; Zhu, Xiangdong

    2013-02-01

    Drug discovery begins by identifying protein-small molecule binding pairs. Afterwards, binding kinetics and biofunctional assays are performed, to reduce candidates for further development. High-throughput screening, typically employing fluorescence, is widely used to find protein ligands in small-molecule libraries, but is rarely used for binding kinetics measurement because: (1) attaching fluorophores to proteins can alter kinetics and (2) most label-free technologies for kinetics measurement are inherently low-throughput and consume expensive sensing surfaces. We addressed this need with polarization-modulated ellipsometric scanning microscopes, called oblique-incidence reflectivity difference (OI-RD). Label-free ligand screening and kinetics measurement are performed simultaneously on small-molecule microarrays printed on relatively inexpensive isocyanate-functionalized glass slides. As a microarray is reacted, an OI-RD microscope tracks the change in surface-bound macromolecule density in real-time at every spot. We report progress applying OI-RD to screen purified proteins and virus particles against a 51,200-compound library from the National Cancer Institute. Four microarrays, each containing 12,800 library compounds, are installed in four flow cells in an automated OI-RD microscope. The slides are reacted serially, each giving 12,800 binding curves with ~30 sec time resolution. The entire library is kinetically screened against a single probe in ~14 hours and multiple probes can be reacted sequentially under automation. Real-time binding detection identifies both high-affinity and low-affinity (transient binding) interactions; fluorescence endpoint images miss the latter. OI-RD and microarrays together is a powerful high-throughput tool for early stage drug discovery and development. The platform also has great potential for downstream steps such as in vitro inhibition assays.

  8. Effects of fasting and semistarvation on the kinetics of active and passive sugar absorption across the small intestine in vivo.

    PubMed Central

    Debnam, E S; Levin, R J

    1975-01-01

    The effects of dietary restriction on the kinetics of absorption in vivo of glucose, galactose and alpha-methyl glucoside were assessed by electrical and chemical methods in the rat jejunum. 2. The 'apparent Km', maximum absorption or Vmax (mu-mole/10 cm. 15 min) and maximum potential difference (p.d.max) were obtained for the jejunal electrogenic active transfer mechanism from the transfer p.d.s and the chemical absorption data corrected for diffusion using various graphical kinetic plots. 3. Fasting for 3 days greatly decreased the 'apparent Kms', obtained from electrical or chemical data, for all the sugars but had no effect on those for L-valine or L-methionine. Semistarvation caused a less pronounced reduction of the 'apparent Kms' for the sugars. The dietary-induced change in 'apparent Km' for glucose was also observed in the fasted hamster. One interpretation of these changes is that the affinity of the carriers for sugars increases during dietary restriction; the greater the level of restriction the greater the increase. 4. Fasting and semistarvation caused large reductions in the Vmax. These reductions were correlated with a reduced enterocyte population estimated by changes in enterocyte column size. 5. The reduction in the Vmax for galactose was mainly accounted for by the decrease in enterocyte population. In the case of glucose, other factors such as reduced enterocyte metabolism or changes in the carriers must be involved to explain the discrepancy between the large decrease in Vmax and the enterocyte column size. 6. Fasting and semi-starvation had complex, differential actions on the p.d.max for glucose, galactose and alpha-methyl glucoside. These changes did not correlate with those observed in the Vmax measured chemically. 7. A standard diet obtained from two commercial sources was found to differ greatly in its effect on the electrogenic transfer system for alpha-methyl glucoside but had no effect on those for galactose and glucose. PMID:1206572

  9. An active learning mammalian skeletal muscle lab demonstrating contractile and kinetic properties of fast- and slow-twitch muscle.

    PubMed

    Head, S I; Arber, M B

    2013-12-01

    The fact that humans possess fast- and slow-twitch muscle in the ratio of ∼50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic properties of fast- and slow-twitch mammalian skeletal muscle. This laboratory illustrates the major differences in contractile properties and fatigue profiles exhibited by the two muscle types. Students compare and contrast twitch kinetics, fused tetanus characteristics, force-frequency relationships, and fatigue properties of fast- and slow-twitch muscles. Examples of results collected by students during class are used to illustrate the type of data collected and analysis performed. During the laboratory, students are encouraged to connect factual information from their skeletal muscle lectures to their laboratory findings. This enables student learning in an active fashion; in particular, the isolated muscle preparation demonstrates that much of what makes muscle fast or slow is myogenic and not the product of the nervous or circulatory systems. This has far-reaching implications for motor control and exercise behavior and therefore is a crucial element in exercise science, with its focus on power and endurance sport activities. To measure student satisfaction with this active learning technique, a questionnaire was administered after the laboratory; 96% of the comments were positive in their support of active versus passive learning strategies.

  10. A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.).

    PubMed

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud'homme, Marie-Pascale; van der Graaff, Eric; Roitsch, Thomas

    2015-01-01

    Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding.

  11. A Simple and Fast Kinetic Assay for the Determination of Fructan Exohydrolase Activity in Perennial Ryegrass (Lolium perenne L.)

    PubMed Central

    Gasperl, Anna; Morvan-Bertrand, Annette; Prud’homme, Marie-Pascale; Roitsch, Thomas

    2015-01-01

    Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding. PMID:26734049

  12. Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell.

    PubMed

    Chen, Yunfeng; Liu, Baoyu; Ju, Lining; Hong, Jinsung; Ji, Qinghua; Chen, Wei; Zhu, Cheng

    2015-08-04

    Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10(-12) N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

  13. Adaptation of acyl-enzyme kinetic theory and an experimental method for evaluating the kinetics of fast-acting, irreversible protease inhibitors.

    PubMed

    Leytus, S P; Peltz, S W; Mangel, W F

    1983-01-26

    The theory of acyl-enzyme kinetics (Bender, M.L., Kézdy, F.J. and Wedler, F.C. (1967) J. Chem. Educ. 44, 84-88) has been adapted for use in evaluating the kinetics of inhibition of serine proteases by both natural and synthetic irreversible inhibitors. The new theory is based upon formal analysis of the case of an irreversible, active-site-directed inhibitor competing with an irreversible, active-site-directed substrate for the active site of a serine protease. From this theory, an experimentally simple and accurate method is described to obtain a second-order rate constant that is characteristic of the efficiency with which an irreversible inhibitor reacts. The experimental method is particularly useful for characterizing fast-acting, irreversible inhibitors. The theory and method which are applicable to a wide variety of enzymes are verified by analysis of the inhibition of bovine trypsin by three model inhibitors, p-nitrophenyl p'-guanidinobenzoate, soybean trypsin inhibitor and alpha-1-proteinase inhibitor as well as by human antithrombin III in the presence of heparin and by bovine pancreatic trypsin inhibitor.

  14. Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics.

    PubMed

    Hartwig, Silvia; Dovengerds, Christine; Herrmann, Christian; Hovemann, Bernhard T

    2014-11-01

    Drosophila Ebony is a β-alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases (NRPSs), which typically consist of at least an adenylation, a peptidyl carrier protein and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to β-alanyl conjugates. If the metabolization of the neurotransmitter histamine to β-alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPSs are in general slowly acting multi-modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remains a key question for understanding its role in vision. To quantitatively analyze the reaction kinetics, we used phosphopantetheinylated holo-Ebony prepared from Baculovirus infected Sf9 cells. Kinetic parameters for the loading reaction, e.g. the formation of β-alanyl-Ebony thioester, complied with those of slow NRPSs. In contrast, single-turnover analysis of the last reaction step, peptide bond formation between pre-activated β-alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This biphasic nature of activity identifies Ebony as a novel type of NRPS related molecule that combines a slow amino acid activation phase with a very fast product formation step.

  15. Disclosure of the oscillations in kinetics of the reactor pressure vessel steel damage at fast neutron intensity decreasing

    NASA Astrophysics Data System (ADS)

    Krasikov, E.; Nikolaenko, V.

    2017-01-01

    Fast neutron intensity influence on reactor materials radiation damage is a critically important question in the problem of the correct use of the accelerated irradiation tests data for substantiation of the materials workability in real irradiation conditions that is low neutron intensity. Investigations of the fast neutron intensity (flux) influence on radiation damage and experimental data scattering reveal the existence of non-monotonous sections in kinetics of the reactor pressure vessels (RPV) steel damage. Discovery of the oscillations as indicator of the self-organization processes presence give reasons for new ways searching on reactor pressure vessel (RPV) steel radiation stability increasing and attempt of the self-restoring metal elaboration. Revealing of the wavelike process in the form of non monotonous parts of the kinetics of radiation embrittlement testifies that periodic transformation of the structure take place. This fact actualizes the problem of more precise definition of the RPV materials radiation embrittlement mechanisms and gives reasons for search of the ways to manage the radiation stability (nanostructuring and so on to stimulate the radiation defects annihilation), development of the means for creating of more stableness self recovering smart materials.

  16. Kinetic analysis of the translocator protein positron emission tomography ligand [(18)F]GE-180 in the human brain.

    PubMed

    Feeney, Claire; Scott, Gregory; Raffel, Joel; Roberts, S; Coello, Christopher; Jolly, Amy; Searle, Graham; Goldstone, A P; Brooks, David J; Nicholas, Richard S; Trigg, William; Gunn, Roger N; Sharp, David J

    2016-11-01

    PET can image neuroinflammation by targeting the translocator protein (TSPO), which is upregulated in activated microglia. The high nonspecific binding of the first-generation TSPO radioligand [(11)C]PK-11195 limits accurate quantification. [(18)F]GE-180, a novel TSPO ligand, displays superior binding to [(11)C]PK-11195 in vitro. Our objectives were to: (1) evaluate tracer characteristics of [(18)F]GE-180 in the brains of healthy human subjects; and (2) investigate whether the TSPO Ala147Thr polymorphism influences outcome measures. Ten volunteers (five high-affinity binders, HABs, and five mixed-affinity binders, MABs) underwent a dynamic PET scan with arterial sampling after injection of [(18)F]GE-180. Kinetic modelling of time-activity curves with one-tissue and two-tissue compartment models and Logan graphical analysis was applied to the data. The primary outcome measure was the total volume of distribution (V T) across various regions of interest (ROIs). Secondary outcome measures were the standardized uptake values (SUV), the distribution volume and SUV ratios estimated using a pseudoreference region. The two-tissue compartment model was the best model. The average regional delivery rate constant (K 1) was 0.01 mL cm(-3) min(-1) indicating low extraction across the blood-brain barrier (1 %). The estimated median V T across all ROIs was also low, ranging from 0.16 mL cm(-3) in the striatum to 0.38 mL cm(-3) in the thalamus. There were no significant differences in V T between HABs and MABs across all ROIs. A reversible two-tissue compartment model fitted the data well and determined that the tracer has a low first-pass extraction (approximately 1 %) and low V T estimates in healthy individuals. There was no observable dependency on the rs6971 polymorphism as compared to other second-generation TSPO PET tracers. Investigation of [(18)F]GE-180 in populations with neuroinflammatory disease is needed to determine its suitability for quantitative assessment

  17. Compartmental analysis of (11C)flumazenil kinetics for the estimation of ligand transport rate and receptor distribution using positron emission tomography

    SciTech Connect

    Koeppe, R.A.; Holthoff, V.A.; Frey, K.A.; Kilbourn, M.R.; Kuhl, D.E. )

    1991-09-01

    The in vivo kinetic behavior of (11C)flumazenil ((11C)FMZ), a non-subtype-specific central benzodiazepine antagonist, is characterized using compartmental analysis with the aim of producing an optimized data acquisition protocol and tracer kinetic model configuration for the assessment of (11C)FMZ binding to benzodiazepine receptors (BZRs) in human brain. The approach presented is simple, requiring only a single radioligand injection. Dynamic positron emission tomography data were acquired on 18 normal volunteers using a 60- to 90-min sequence of scans and were analyzed with model configurations that included a three-compartment, four-parameter model, a three-compartment, three-parameter model, with a fixed value for free plus nonspecific binding; and a two-compartment, two-parameter model. Statistical analysis indicated that a four-parameter model did not yield significantly better fits than a three-parameter model. Goodness of fit was improved for three- versus two-parameter configurations in regions with low receptor density, but not in regions with moderate to high receptor density. Thus, a two-compartment, two-parameter configuration was found to adequately describe the kinetic behavior of (11C)FMZ in human brain, with stable estimates of the model parameters obtainable from as little as 20-30 min of data. Pixel-by-pixel analysis yields functional images of transport rate (K1) and ligand distribution volume (DV), and thus provides independent estimates of ligand delivery and BZR binding.

  18. Unchanged content of oxidative enzymes in fast-twitch muscle fibers and kinetics after intensified training in trained cyclists

    PubMed Central

    Christensen, Peter M; Gunnarsson, Thomas P; Thomassen, Martin; Wilkerson, Daryl P; Nielsen, Jens Jung; Bangsbo, Jens

    2015-01-01

    The present study examined if high intensity training (HIT) could increase the expression of oxidative enzymes in fast-twitch muscle fibers causing a faster oxygen uptake () response during intense (INT), but not moderate (MOD), exercise and reduce the slow component and muscle metabolic perturbation during INT. Pulmonary kinetics was determined in eight trained male cyclists (-max: 59 ± 4 (means ± SD) mL min−1 kg−1) during MOD (205 ± 12 W ∼65% -max) and INT (286 ± 17 W ∼85% -max) exercise before and after a 7-week HIT period (30-sec sprints and 4-min intervals) with a 50% reduction in volume. Both before and after HIT the content in fast-twitch fibers of CS (P < 0.05) and COX-4 (P < 0.01) was lower, whereas PFK was higher (P < 0.001) than in slow-twitch fibers. Content of CS, COX-4, and PFK in homogenate and fast-twitch fibers was unchanged with HIT. Maximal activity (μmol g DW−1 min−1) of CS (56 ± 8 post-HIT vs. 59 ± 10 pre-HIT), HAD (27 ± 6 vs. 29 ± 3) and PFK (340 ± 69 vs. 318 ± 105) and the capillary to fiber ratio (2.30 ± 0.16 vs. 2.38 ± 0.20) was unaltered following HIT. kinetics was unchanged with HIT and the speed of the primary response did not differ between MOD and INT. Muscle creatine phosphate was lower (42 ± 15 vs. 66 ± 17 mmol kg DW−1) and muscle lactate was higher (40 ± 18 vs. 14 ± 5 mmol kg DW−1) at 6 min of INT (P < 0.05) after compared to before HIT. A period of intensified training with a volume reduction did not increase the content of oxidative enzymes in fast-twitch fibers, and did not change kinetics. PMID:26152692

  19. Kinetic effects in the conversion of fast waves in pre-heated, low aspect ratio tokamak plasmas

    NASA Astrophysics Data System (ADS)

    Kommoshvili, K.; Cuperman, S.; Bruma, C.

    2003-03-01

    Kinetic effects in the conversion of fast waves to Alfvèn waves and their subsequent deposition in low aspect ratio (spherical) tokamaks (LARTs) have been investigated theoretically. More specifically, we have considered the consequences of incorporation of kinetic effects in the electron parallel (to the ambient magnetic field) dynamics derived by following the drift-tearing mode analysis of Chen et al (Chen L, Rutherford P H and Tang W M 1977 Phys. Rev. Lett. 39 460), and particle-conserving Krook collision operator for the passing electrons involved (Mett R R and Mahajan S M 1992 Phys. Fluids B 4 2885). The perpendicular plasma dynamics is described by a quite general resistive two-fluid (2F) model based dielectric tensor-operator (Cuperman S, Bruma C and Komoshvili K 2002 Solution of the resistive 2F wave equations for Alfvènic modes in spherical tokamak plasmas J. Plasma Phys. accepted for publication). The full-wave electromagnetic equations, formulated in terms of the vector and scalar potentials, have been solved by the aid of an advanced finite elements numerical code (Sewell G 1993 Adv. Eng. Software 17 105). Detailed solutions of the full-wave equations are obtained and compared with those corresponding to a pure resistive 2F model, this, for the illustrative pre-heated START-type device (Sykes 1994). Our results quantitatively confirm the general theory of the conversion of fast waves with subsequent power dissipation for the conditions of spherical tokamaks thus providing the required auxilliary energy source for the succesful operation of LARTs. Moreover, these results indicate the absolute necessity of using a full model for the parallel electron dynamics, i.e. including both kinetic and collisional effects.

  20. Efficient removal of organic ligands from supported nanocrystals by fast thermal annealing enables catalytic studies on well-defined active phases.

    PubMed

    Cargnello, Matteo; Chen, Chen; Diroll, Benjamin T; Doan-Nguyen, Vicky V T; Gorte, Raymond J; Murray, Christopher B

    2015-06-03

    A simple yet efficient method to remove organic ligands from supported nanocrystals is reported for activating uniform catalysts prepared by colloidal synthesis procedures. The method relies on a fast thermal treatment in which ligands are quickly removed in air, before sintering can cause changes in the size and shape of the supported nanocrystals. A short treatment at high temperatures is found to be sufficient for activating the systems for catalytic reactions. We show that this method is widely applicable to nanostructures of different sizes, shapes, and compositions. Being rapid and effective, this procedure allows the production of monodisperse heterogeneous catalysts for studying a variety of structure-activity relationships. We show here results on methane steam reforming, where the particle size controls the CO/CO2 ratio on alumina-supported Pd, demonstrating the potential applications of the method in catalysis.

  1. Development of a Fast Microfluidic Mixer for Studies of Protein Folding KineticsFinal Report Cover Page

    SciTech Connect

    Bakajin, O

    2005-02-10

    We designed and fabricated mixing devices that will help us elucidate the mechanisms of protein folding through measurements of folding reaction rates. These devices can be used in studying of other biological systems and are compatible with various spectroscopic observation methods. The project involved development of fabrication processes and setup of a laboratory for assembly and characterization of microfluidic devices, as well as measurements of protein folding kinetics. We produced three variants of the mixer: (1) The ultra fast mixer for Foerster Resonance Energy Transfer measurements (described by Anal. Chem. Article UCRL-JRNL-206676) and MicroTAS Conference Proceedings article (UCRL-JC-153057 ) included in the report; (2) The ultra fast mixer for UV measurements (described by the poster presented at MicroTAS conference (UCRL-POST-207476) included in the report); and (3) The mixer for single molecule measurements (described by the Science article UCRL-JC-153057) included in the report. In these mixers, the channels are narrow, ranging from a few to hundreds of {micro}m, so that the flow is laminar and all of the mixing is achieved through diffusion. Our goal is to develop robust microfluidic mixer with at least 100 times lower consumption rate, shorter dead time and time resolution than commercially available mixers that would be compatible with most commonly used spectroscopic methods. We are also developing mixers that can be used in combination with single molecule spectroscopy. The mixers are used to study kinetics of fast protein folding reactions using bulk fluorescence and single molecule fluorescence resonance energy transfer techniques. Capabilities for microfluidic have been developed at BSNL that will be useful for studies of interactions of DNA with proteins and other projects such as the single molecule detector for detection of low concentration of toxins.

  2. The superfast human extraocular myosin is kinetically distinct from the fast skeletal IIa, IIb, and IId isoforms.

    PubMed

    Bloemink, Marieke J; Deacon, John C; Resnicow, Daniel I; Leinwand, Leslie A; Geeves, Michael A

    2013-09-20

    Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C2C12 muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K(AD)) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K(AD) values of 118, 80, and 55 μM for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K(AD) = 352 μM. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.

  3. Preliminary results of fasting on the kinetics of organochlorines in polar bears (Ursus maritimus).

    PubMed

    Polischuk, S C; Letcher, R J; Norstrom, R J; Ramsay, M A

    1995-01-15

    We determined concentrations of polychlorinated biphenyls (PCBs), chlordanes (CHLORs), chlorobenzenes (CBzs), hexachlorocyclohexanes (HCHs), and dichlorodiphenyltrichloroethane (DDT) and its metabolites (DDD and DDE) in the tissues of individual polar bears (Ursus maritimus) before and after a lengthy period of fasting. Polar bears are an ideal model for such studies. They undergo one of the most extreme fasts known for any mammal and are located at the top of a long food chain, thus biomagnification of organochlorines (OC) is significant. Adipose tissue and milk were collected from different reproductive classes of adult females (solitary/pregnant, with cubs-of-the-year, with yearling cubs) and were analyzed for organochlorine content. As the fasting period progressed and adipose reserves decreased, concentrations of some organochlorines in the adipose tissue and milk increased on a lipid weight basis. The transfer of contaminants from mothers to offspring thus also increases with duration of the fasting period. This phenomenon could adversely influence the survival and growth of cubs during the critical early phase of their development.

  4. Generalized Temporal Acceleration Scheme for Kinetic Monte Carlo Simulations of Surface Catalytic Processes by Scaling the Rates of Fast Reactions.

    PubMed

    Dybeck, Eric Christopher; Plaisance, Craig Patrick; Neurock, Matthew

    2017-02-14

    A novel algorithm has been developed to achieve temporal acceleration during kinetic Monte Carlo (KMC) simulations of surface catalytic processes. This algorithm allows for the direct simulation of reaction networks containing kinetic processes occurring on vastly disparate timescales which computationally overburden standard KMC methods. Previously developed methods for temporal acceleration in KMC have been designed for specific systems and often require a priori information from the user such as identifying the fast and slow processes. In the approach presented herein, quasi-equilibrated processes are identified automatically based on previous executions of the forward and reverse reactions. Temporal acceleration is achieved by automatically scaling the intrinsic rate constants of the quasi-equilibrated processes, bringing their rates closer to the timescales of the slow kinetically relevant non-equilibrated processes. All reactions are still simulated directly, although with modified rate constants. Abrupt changes in the underlying dynamics of the reaction network are identified during the simulation and the reaction rate constants are rescaled accordingly. The algorithm has been utilized here to model the Fischer-Tropsch synthesis reaction over ruthenium nanoparticles. This reaction network has multiple timescale-disparate processes which would be intractable to simulate without the aid of temporal acceleration. The accelerated simulations are found to give reaction rates and selectivities indistinguishable from those calculated by an equivalent mean-field kinetic model. The computational savings of the algorithm can span many orders of magnitude in realistic systems and the computational cost is not limited by the magnitude of the timescale disparity in the system processes. Furthermore, the algorithm has been designed in a generic fashion and can easily be applied to other surface catalytic processes of interest.

  5. Influence of composition on setting kinetics of new injectable and/or fast setting tricalcium silicate cements.

    PubMed

    Setbon, H M; Devaux, J; Iserentant, A; Leloup, G; Leprince, J G

    2014-12-01

    New commercial tricalcium silicate based cements were elaborated to improve handling properties and setting time. The goals of the present work were: (i) to determine the composition of the new injectable and/or fast setting calcium silicate based cements, and (ii) to investigate the impact of the differences in composition on their setting kinetics. The materials considered were Angelus MTA™, Biodentine™, MM-MTA™, MTA-Caps™, and ProRoot MTA™ as control. Elemental composition of materials was studied by Inductively Coupled Plasma-Atomic Emission Spectroscopy and X-ray Energy Dispersive analysis, whereas phases in presence were analyzed by Micro-Raman spectroscopy and X-ray Diffraction analysis and cement surface by Scanning Electron Microscope. Setting kinetics was evaluated using rheometry. Elemental analysis revealed, for all cements, the presence of three major components: calcium, silicon and oxygen. Chlorine was detected in MM-MTA, MTA-Caps and Biodentine. Different radio-opacifiers were identified: bismuth oxide in ProRoot MTA, Angelus MTA and MM-MTA, zirconium oxide in Biodentine and calcium tungstate (CaWO4) in MTA-Caps. All cements were composed of di- and tri-calcium silicate, except Biodentine for which only the latter was detected. Major differences in setting kinetics were observed: a modulus of 8×10(8)Pa is reached after 12min for Biodentine, 150min for MM-MTA, 230min for Angelus MTA and 320min for ProRoot MTA. The maximum modulus reached by MTA-Caps was 7×10(8)Pa after 150min. Even if these cements possess some common compounds, major differences in their composition were observed between them, which directly influence their setting kinetics. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  6. Edge-bridging and face-capping coordination of alkenyl ligands in triruthenium carbonyl cluster complexes derived from hydrazines: synthetic, structural, theoretical, and kinetic studies.

    PubMed

    Cabeza, Javier A; del Río, Ignacio; Fernández-Colinas, José M; García-Granda, Santiago; Martínez-Méndez, Lorena; Pérez-Carreño, Enrique

    2004-12-03

    The reactions of the triruthenium cluster complex [Ru3(mu-H)(mu3-eta2-HNNMe2)(CO)9] (1; H2NNMe2=1,1-dimethylhydrazine) with alkynes (PhC triple bond CPh, HC triple bond CH, MeO2CC triple bond CCO2Me, PhC triple bond CH, MeO2CC triple bond CH, HOMe2CC triple bond CH, 2-pyC triple bond CH) give trinuclear complexes containing edge-bridging and/or face-capping alkenyl ligands. Whereas the edge-bridged products are closed triangular species (three Ru-Ru bonds), the face-capped products are open derivatives (two Ru-Ru bonds). For terminal alkynes, products containing gem (RCCH2) and/or trans (RHCCH) alkenyl ligands have been identified in both edge-bridging and face-capping positions, except for the complex [Ru3(mu3-eta2-HNNMe2)(mu3-eta3-HCCH-2-py)(mu-CO)(CO)7], which has the two alkenyl H atoms in a cis arrangement. Under comparable reaction conditions (1:1 molar ratio, THF at reflux, time required for the consumption of complex 1), some reactions give a single product, but most give mixtures of isomers (not all the possible ones), which were separated. To determine the effect of the hydrazido ligand, the reactions of [Ru3(mu-H)(mu3-eta2-MeNNHMe)(CO)9] (2; HMeNNHMe=1,2-dimethylhydrazine) with PhC triple bond CPh, PhC triple bond CH, and HC triple bond CH were also studied. For edge-bridged alkenyl complexes, the Ru--Ru edge that is spanned by the alkenyl ligand depends on the position of the methyl groups on the hydrazido ligand. For face-capped alkenyl complexes, the relative orientation of the hydrazido and alkenyl ligands also depends on the position of the methyl groups on the hydrazido ligand. A kinetic analysis of the reaction of 1 with PhC[triple chemical bond]CPh revealed that the reaction follows an associative mechanism, which implies that incorporation of the alkyne in the cluster is rate-limiting and precedes the release of a CO ligand. X-ray diffraction, IR and NMR spectroscopy, and calculations of minimum-energy structures by DFT methods were used to

  7. Mg-based nanocomposites with high capacity and fast kinetics for hydrogen storage.

    PubMed

    Yao, Xiangdong; Wu, Chengzhang; Du, Aijun; Lu, Gao Qing; Cheng, Huiming; Smith, Sean C; Zou, Jin; He, Yinghe

    2006-06-22

    Magnesium and its alloys have shown a great potential in effective hydrogen storage due to their advantages of high volumetric/gravimetric hydrogen storage capacity and low cost. However, the use of these materials in fuel cells for automotive applications at the present time is limited by high hydrogenation temperature and sluggish sorption kinetics. This paper presents the recent results of design and development of magnesium-based nanocomposites demonstrating the catalytic effects of carbon nanotubes and transition metals on hydrogen adsorption in these materials. The results are promising for the application of magnesium materials for hydrogen storage, with significantly reduced absorption temperatures and enhanced ab/desorption kinetics. High level Density Functional Theory calculations support the analysis of the hydrogenation mechanisms by revealing the detailed atomic and molecular interactions that underpin the catalytic roles of incorporated carbon and titanium, providing clear guidance for further design and development of such materials with better hydrogen storage properties.

  8. Site specific ligand substitution in cubane-type Mo3FeS(4)(4+) clusters: kinetics and mechanism of reaction and isolation of mixed ligand Cl/SPh complexes.

    PubMed

    Algarra, Andrés G; Basallote, Manuel G; Fernandez-Trujillo, M J; Llusar, Rosa; Pino-Chamorro, Jose A; Sorribes, Ivan; Vicent, Cristian

    2010-04-21

    The synthesis, crystal structure and solution characterization of the cubane-type [Mo(3)(FeCl)S(4)(dmpe)(3)Cl(3)] (1) (dmpe = 1,2-bis(dimethylphophane-ethane)) cluster are reported and the ligand substitution processes of chloride by thiophenolate investigated. The kinetics and the intimate mechanism of these substitutions reveal that compound 1 undergoes a number of Fe and Mo site specific ligand substitution reactions in acetonitrile solutions. In particular, PhS(-) coordination at the tetrahedral Fe site proceeds in a single resolved kinetic step whereas such substitutions at the Mo sites proceed more slowly. The effect of the presence of acids in the reaction media is also investigated and reveals that an acid excess hinders substitution reactions both at the Fe and Mo sites; however, an acid-promoted solvolysis of the Fe-Cl bonds is observed. Electrospray ionization (ESI) and tandem (ESI-MS/MS) mass spectrometry allow the identification of all the reaction intermediates proposed on the basis of stopped-flow measurements. The distinctive site specific reactivity made it possible to isolate two new clusters of the Mo(3)FeS(4)(4+) family featuring mixed chlorine/thiophenolate ligands, namely Mo(3)S(4)(FeSPh)(dmpe)(3)Cl(3) (2) and [Mo(3)S(4)(FeSPh)(dmpe)(3)(SPh)(3)] (3). A detailed computational study has also been carried out to understand the details of the mechanism of substitution at the M-Cl (M = Mo and Fe) bonds as well as the solvolysis at the Fe-Cl sites, with particular emphasis on the role of acids on the substitution process. The results of the calculations are in agreement with the experimental observations, thus justifying the non-existence of an accelerating effect of acids on the thiophenolate substitution reaction, which differs from previous proposals for the Fe(4)S(4) and MoFe(3)S(4) clusters and some related compounds.

  9. Characterizing the micro structure and kinetics of fast changing samples by simultaneous polarization measurements

    NASA Astrophysics Data System (ADS)

    Liao, Ran; He, Honghui; Zeng, Nan; Ma, Hui

    2015-03-01

    Taking accurate measurements of the state of polarization (SOP) is the key for the success of polarization sensitive techniques which can provide rich information on the microstructure of complex scattering media, such as biological tissues. For static or slow varying samples, SOP measurements can be achieved by time-sequential recoding of different polarization components controlled by rotating polarizers and wave plates or temporal modulation devices such as photoelastic modulators or liquid crystal variable retarders. When the sample is moving or changing its status quickly, polarization components recoded at different time may correspond to different SOPs, which can lead to significant errors in the final results. Simultaneous polarization measurements are necessary for probing such dynamic samples. In this paper, using the simultaneously recorded polarization components, we are able to mimic time sequential polarization schemes and evaluate the errors. The results show that the kinetics of the sample will affect the systematic error and an increase in the statistical errors of the measured degree of polarization (DOP). We change the kinetics of samples with different stirring speed, which is indicated by the characteristic time of the auto-correlation function. It is also demonstrated that the simultaneously recorded polarization components reveals additional information on the orientation of fibrous scatterers as well as their translation and rotation kinetics.

  10. KID - an algorithm for fast and efficient text mining used to automatically generate a database containing kinetic information of enzymes

    PubMed Central

    2010-01-01

    Background The amount of available biological information is rapidly increasing and the focus of biological research has moved from single components to networks and even larger projects aiming at the analysis, modelling and simulation of biological networks as well as large scale comparison of cellular properties. It is therefore essential that biological knowledge is easily accessible. However, most information is contained in the written literature in an unstructured way, so that methods for the systematic extraction of knowledge directly from the primary literature have to be deployed. Description Here we present a text mining algorithm for the extraction of kinetic information such as KM, Ki, kcat etc. as well as associated information such as enzyme names, EC numbers, ligands, organisms, localisations, pH and temperatures. Using this rule- and dictionary-based approach, it was possible to extract 514,394 kinetic parameters of 13 categories (KM, Ki, kcat, kcat/KM, Vmax, IC50, S0.5, Kd, Ka, t1/2, pI, nH, specific activity, Vmax/KM) from about 17 million PubMed abstracts and combine them with other data in the abstract. A manual verification of approx. 1,000 randomly chosen results yielded a recall between 51% and 84% and a precision ranging from 55% to 96%, depending of the category searched. The results were stored in a database and are available as "KID the KInetic Database" via the internet. Conclusions The presented algorithm delivers a considerable amount of information and therefore may aid to accelerate the research and the automated analysis required for today's systems biology approaches. The database obtained by analysing PubMed abstracts may be a valuable help in the field of chemical and biological kinetics. It is completely based upon text mining and therefore complements manually curated databases. The database is available at http://kid.tu-bs.de. The source code of the algorithm is provided under the GNU General Public Licence and available on

  11. Laser Temperature-Jump Kinetic Studies of Fast Reactions in Nonaqueous Solutions

    DTIC Science & Technology

    1990-02-28

    Rose, E. N. Eyring, L. J. Rodriguez, and S. Petrucci , J. Phys. Chem. 21, 1396 (1989). 2. L. J. Rodriguez, M. Xu, E. M. Eyring and S. Petrucci , Pure...Appl. Chem. §1 , 1593 (1989). 3. J. Eschmann, J. Strasser, M. Xu, Y. Okamoto, E. H. Eyring and S. Petrucci , J. Phys. Chem., in press. 4. B. A...Barton, D. J. Rose, E. H. Eyring, L. J. Rodriguez, and S. Petrucci , "Kinetics of Complexation of Lithium Perchlorate with 18-Crown-6 in Propylene

  12. The derivative assay--an analysis of two fast components of DNA rejoining kinetics

    SciTech Connect

    Sandstroem, B.E. )

    1989-11-01

    The DNA rejoining kinetics of human U-118 MG cells were studied after gamma-irradiation with 4 Gy. The analysis of the sealing rate of the induced DNA strand breaks was made with a modification of the DNA unwinding technique. The modification meant that rather than just monitoring the number of existing breaks at each time of analysis, the velocity, at which the rejoining process proceeded, was determined. Two apparent first-order components of single-strand break repair could be identified during the 25 min of analysis. The half-times for the two components were 1.9 and 16 min, respectively.

  13. Comparison of the kinetic performance of different columns for fast liquid chromatography, emphasizing the contributions of column end structure.

    PubMed

    Lambert, Nándor; Miyazaki, Shota; Ohira, Masayoshi; Tanaka, Nobuo; Felinger, Attila

    2016-11-18

    The kinetic performance of five chromatographic columns designed for fast liquid chromatography with different column packing materials - including fully porous (2.0 and 1.9μm particles), core-shell (2.6μm particles) or monolithic packings - with identical column dimensions (2.1×50mm) was tested. Since the tested monolithic column showed systematically better efficiency for early eluting compounds than the packed columns, an additional band broadening effect was suspected for the packed columns. The effects of the presence of the frits and the bed heterogeneity of the columns near the frits were characterized by a column-reversal method. It has been shown that significant differences - even 20-25% difference in efficiency - can exist between the two ends of the packed columns, while the monolithic column shows rather similar performance at either column end.

  14. Kinetic modelling indicates that fast-translating codons can coordinate cotranslational protein folding by avoiding misfolded intermediates

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward P.; Vendruscolo, Michele; Dobson, Christopher M.

    2014-01-01

    It has been observed for several proteins that slowing down the rate at which individual codons are translated can increase their probability of cotranslational protein folding, while speeding up codon translation can decrease it. Here we investigate whether or not this inverse relationship between translation speed and the cotranslational folding probability is a general phenomenon or if other scenarios are possible. We first derive chemical kinetic equations that relate individual codon translation rates to the probability that a domain will fold, populate an intermediate or misfold, and examine the cotranslational folding scenarios that are possible within these models. We find that speeding up codon translation through misfolding-prone segments can, in some cases, increase the folding probability of a domain immediately before the nascent protein is released from the ribosome and decrease its chances of misfolding. Thus, for some proteins fast-translating codons could be as important as slow-translating codons in coordinating cotranslational protein folding.

  15. Photoreductive dissolution of iron(III) (hydr)oxides in the absence and presence of organic ligands: experimental studies and kinetic modeling.

    PubMed

    Borer, Paul; Sulzberger, Barbara; Hug, Stephan J; Kraemer, Stephan M; Kretzschmar, Ruben

    2009-03-15

    This study investigated the kinetics of the photoreductive dissolution of various iron(III)(hydr)oxide phases, lepidocrocite (gamma-FeOOH), ferrihydrite, and hydrous ferric oxide, in the absence of organic ligands as a function of pH in deaerated and aerated suspensions. Photoreductive dissolution of lepidocrocite and ferrihydrite only occurred below pH 6. Under oxic conditions, we observed both the formation of aqueous Fe(II) and H2O2 during photoreductive dissolution of lepidocrocite and ferrihydrite at pH 3. These experimental findings are consistent with the light-induced reduction of surface Fe(III) at the (hydr)oxide surface and the concomitant oxidation of surface-coordinated water or hydroxyl groups, leading to surface Fe(II) and *OH radicals and subsequently to H2O2. The formation of *OH radicals atthe surface was confirmed by photodissolution experiments conducted in the presence of *OH radical scavengers. Kinetic modeling of the experimental data suggests that the relevant pathway for the formation of H2O2 is the reoxidation of surface lattice Fe(II) by molecular oxygen. This study furthermore shows that in the presence of strong iron binding ligands such as siderophores, specifically desferrioxamine B, the photoreductive dissolution of lepidocrocite, ferrihydrite, and to a lesser extent hydrous ferric oxide may also proceed at seawater pH.

  16. Frequency-Dependent Modulation of Dopamine Release by Nicotine and Dopamine D1 Receptor Ligands: An In Vitro Fast Cyclic Voltammetry Study in Rat Striatum.

    PubMed

    Goutier, W; Lowry, J P; McCreary, A C; O'Connor, J J

    2016-05-01

    Nicotine is a highly addictive drug and exerts this effect partially through the modulation of dopamine release and increasing extracellular dopamine in regions such as the brain reward systems. Nicotine acts in these regions on nicotinic acetylcholine receptors. The effect of nicotine on the frequency dependent modulation of dopamine release is well established and the purpose of this study was to investigate whether dopamine D1 receptor (D1R) ligands have an influence on this. Using fast cyclic voltammetry and rat corticostriatal slices, we show that D1R ligands are able to modulate the effect of nicotine on dopamine release. Nicotine (500 nM) induced a decrease in dopamine efflux at low frequency (single pulse or five pulses at 10 Hz) and an increase at high frequency (100 Hz) electrical field stimulation. The D1R agonist SKF-38393, whilst having no effect on dopamine release on its own or on the effect of nicotine upon multiple pulse evoked dopamine release, did significantly prevent and reverse the effect of nicotine on single pulse dopamine release. Interestingly similar results were obtained with the D1R antagonist SCH-23390. In this study we have demonstrated that the modulation of dopamine release by nicotine can be altered by D1R ligands, but only when evoked by single pulse stimulation, and are likely working via cholinergic interneuron driven dopamine release.

  17. Competitive binding-based optical DNA mapping for fast identification of bacteria--multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

    PubMed

    Nilsson, Adam N; Emilsson, Gustav; Nyberg, Lena K; Noble, Charleston; Stadler, Liselott Svensson; Fritzsche, Joachim; Moore, Edward R B; Tegenfeldt, Jonas O; Ambjörnsson, Tobias; Westerlund, Fredrik

    2014-09-01

    We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli

    PubMed Central

    Nilsson, Adam N.; Emilsson, Gustav; Nyberg, Lena K.; Noble, Charleston; Stadler, Liselott Svensson; Fritzsche, Joachim; Moore, Edward R. B.; Tegenfeldt, Jonas O.; Ambjörnsson, Tobias; Westerlund, Fredrik

    2014-01-01

    We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens. PMID:25013180

  19. Pd-catalyzed amidation of aryl chlorides using monodentate biaryl phosphine ligands: a kinetic, computational, and synthetic investigation.

    PubMed

    Ikawa, Takashi; Barder, Timothy E; Biscoe, Mark R; Buchwald, Stephen L

    2007-10-31

    We present results on the amidation of aryl halides and sulfonates utilizing a monodentate biaryl phosphine-Pd catalyst. Our results are in accord with a previous report that suggests that the formation of kappa(2)-amidate complexes is deleterious to the effectiveness of a catalyst for this transformation and that their formation can be prevented by the use of appropriate bidentate ligands. We now provide data that suggest that the use of certain monodentate ligands can also prevent the formation of the kappa(2)-amidate complexes and thereby generate more stable catalysts for the amination of aryl chlorides. Furthermore, computational studies shed light on the importance of the key feature(s) of the biaryl phosphines (a methyl group ortho to the phosphorus center) that enable the coupling to occur. The use of ligands that possess a methyl group ortho to the phosphorus center allows a variety of aryl and heteroaryl chlorides with various amides to be coupled in high yield.

  20. Differential kinetics of fast and slow ankle extensors during the paw-shake in the cat.

    PubMed

    Fowler, E G; Gregor, R J; Roy, R R

    1988-01-01

    Force, length, and EMG were assessed in the medial gastrocnemius and soleus muscles of two cats during the paw-shake response. The medial gastrocnemius produced high forces and significant electrical activity while force production and electrical activity were negligible in the soleus. This observation is significant as it provides evidence, through the direct measurement of muscle force, of selective recruitment of a fast muscle when a slow synergist is not activated. Additionally, the relationship among force, length, and neural activation indicates that the role of the medial gastrocnemius during the paw-shake response is to decelerate muscle lengthening and begin muscle shortening.

  1. Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands.

    PubMed Central

    Stevenson, T H; Gutierrez, A F; Alderton, W K; Lian , L; Scrutton, N S

    2001-01-01

    The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) cofactor L-arginine and several Type I L-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH(4) in the iNOS active site has little effect on the rebinding of protein-caged haem-CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of L-arginine to the BH(4)-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of L-arginine or BH(4). In the presence of BH(4) and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes. PMID:11485568

  2. Environmentally-friendly aqueous Li (or Na)-ion battery with fast electrode kinetics and super-long life

    PubMed Central

    Dong, Xiaoli; Chen, Long; Liu, Jingyuan; Haller, Servane; Wang, Yonggang; Xia, Yongyao

    2016-01-01

    Current rechargeable batteries generally display limited cycle life and slow electrode kinetics and contain environmentally unfriendly components. Furthermore, their operation depends on the redox reactions of metal elements. We present an original battery system that depends on the redox of I−/I3− couple in liquid cathode and the reversible enolization in polyimide anode, accompanied by Li+ (or Na+) diffusion between cathode and anode through a Li+/Na+ exchange polymer membrane. There are no metal element–based redox reactions in this battery, and Li+ (or Na+) is only used for charge transfer. Moreover, the components (electrolyte/electrode) of this system are environment-friendly. Both electrodes are demonstrated to have very fast kinetics, which gives the battery a supercapacitor-like high power. It can even be cycled 50,000 times when operated within the electrochemical window of 0 to 1.6 V. Such a system might shed light on the design of high-safety and low-cost batteries for grid-scale energy storage. PMID:26844298

  3. Environmentally-friendly aqueous Li (or Na)-ion battery with fast electrode kinetics and super-long life.

    PubMed

    Dong, Xiaoli; Chen, Long; Liu, Jingyuan; Haller, Servane; Wang, Yonggang; Xia, Yongyao

    2016-01-01

    Current rechargeable batteries generally display limited cycle life and slow electrode kinetics and contain environmentally unfriendly components. Furthermore, their operation depends on the redox reactions of metal elements. We present an original battery system that depends on the redox of I(-)/I3 (-) couple in liquid cathode and the reversible enolization in polyimide anode, accompanied by Li(+) (or Na(+)) diffusion between cathode and anode through a Li(+)/Na(+) exchange polymer membrane. There are no metal element-based redox reactions in this battery, and Li(+) (or Na(+)) is only used for charge transfer. Moreover, the components (electrolyte/electrode) of this system are environment-friendly. Both electrodes are demonstrated to have very fast kinetics, which gives the battery a supercapacitor-like high power. It can even be cycled 50,000 times when operated within the electrochemical window of 0 to 1.6 V. Such a system might shed light on the design of high-safety and low-cost batteries for grid-scale energy storage.

  4. In situ and in silico kinetic analyses of the Programmed Cell Death 1, Programmed Cell Death-Ligands, and B7-1 interaction network.

    PubMed

    Li, Kaitao; Cheng, Xiaoxiao; Tilevik, Andreas; Davis, Simon J; Zhu, Cheng

    2017-03-06

    Programmed cell death-1 (PD-1) is an inhibitory receptor with an essential role in maintaining peripheral tolerance, and among the most promising immunotherapeutic targets for treating cancer, autoimmunity, and infectious diseases. A complete understanding of the consequences of PD-1 engagement by its ligands, PD-L1 and PD-L2, and of PD-L1 binding to B7-1, requires quantitative analysis of their interactions at the cell surface. We present here the first complete in situ kinetic analysis of the PD-1/PD-1 ligands/B7-1 system. Consistent with previous solution measurements, we observed higher in situ affinities for human (h) than murine (m) PD-1 interactions, stronger binding of hPD-1 to hPD-L2 than hPD-L1, and comparable binding of mPD-1 to both ligands. However, in contrast to the relatively weak solution affinities, the in situ affinities of PD-1 are as high as those of the TCR for agonist pMHC and of LFA-1 for ICAM-1, but significantly lower than that of the B7-1-CTLA-4 interaction, suggesting a distinct basis for PD-1 versus CTLA-4 mediated inhibition. Notably, the in situ interactions of PD-1 are much stronger than that of B7-1 with PD-L1. Overall, the in situ affinity ranking greatly depends on the on-rate instead of the off-rate. In silico simulations predict that PD-1-PD-L1 interactions dominate at interfaces between activated T cells and mature dendritic cells, and that these interactions will be highly sensitive to the dynamics of PD-L1 and PD-L2 expression. Our results provide a kinetic framework for better understanding inhibitory PD-1 activity in health and disease.

  5. Fast and slow crystal growth kinetics in glass-forming melts

    NASA Astrophysics Data System (ADS)

    Orava, J.; Greer, A. L.

    2014-06-01

    Published values of crystal growth rates are compared for supercooled glass-forming liquids undergoing congruent freezing at a planar crystal-liquid interface. For the purposes of comparison pure metals are considered to be glass-forming systems, using data from molecular-dynamics simulations. For each system, the growth rate has a maximum value Umax at a temperature Tmax that lies between the glass-transition temperature Tg and the melting temperature Tm. A classification is suggested, based on the lability (specifically, the propensity for fast crystallization), of the liquid. High-lability systems show "fast" growth characterized by a high Umax, a low Tmax / Tm, and a very broad peak in U vs. T / Tm. In contrast, systems showing "slow" growth have a low Umax, a high Tmax / Tm, and a sharp peak in U vs. T / Tm. Despite the difference of more than 11 orders of magnitude in Umax seen in pure metals and in silica, the range of glass-forming systems surveyed fit into a common pattern in which the lability increases with lower reduced glass-transition temperature (Tg / Tm) and higher fragility of the liquid. A single parameter, a linear combination of Tg / Tm and fragility, can show a good correlation with Umax. For all the systems, growth at Umax is coupled to the atomic/molecular mobility in the liquid. It is found that, across the diversity of glass-forming systems, Tmax / Tg = 1.48 ± 0.15.

  6. Nanoporous Au-Ag shell with fast kinetics: integrating chemical and plasmonic catalysis.

    PubMed

    Yang, Wenpeng; Wu, Ke; Yang, Weimin; Wang, Hailing; Lv, Xianglong; Qian, Lihua; Yu, Tianshui; Li, Zengquan; Zhou, Xiangji; Barasa, Godfrey Okumu; Yuan, S L; Jiang, Yingtao; Yang, Zhilin

    2017-08-07

    Nanoporous noble metals and alloys are widely utilized as efficient catalysts, because they have high surface-to-volume ratios for surfficient active sites and induce molecule polarization through plasmon excitation as well. Herein we demonstrate one approach to fabricate nanoporous Au-Ag shell. Such material represents the dual functions serving as efficient catalysts and high-performance surface-enhanced Raman scattering (SERS) substrate. In-situ spectrum acquisition can track the conversion of p-nitrothiophenol to 4, 4'-dimercapto-azobenzene at ambient temperature. In particular, as a result of chemcial catalysis of Ag elements and strong plasmon-molecule coupling, catalytic kinetics of nanoporous Au-Ag shell is 79.2-123.8 times faster than Au nanoparticles, and 2.2-3.3 times faster than Ag nanoparticles. This investigation offers a route to design superior catalysts to integrate chemical and plasmonic catalysis. © 2017 IOP Publishing Ltd.

  7. Nanoporous Au-Ag shell with fast kinetics: integrating chemical and plasmonic catalysis

    NASA Astrophysics Data System (ADS)

    Yang, Wenpeng; Wu, Ke; Yang, Weimin; Wang, Hailing; Lv, Xianglong; Qian, Lihua; Yu, Tianshui; Li, Zengquan; Zhou, Xiangji; Okumu Barasa, Godfrey; Yuan, Songliu; Jiang, Yingtao; Yang, Zhilin

    2017-10-01

    Nanoporous noble metals and alloys are widely utilized as efficient catalysts, because they have high surface-to-volume ratios for sufficient active sites and induce molecule polarization through plasmon excitation as well. Herein, we demonstrate one approach to fabricate nanoporous Au-Ag shell. Such material represents the dual functions serving as efficient catalysts and high-performance surface-enhanced Raman scattering substrate. In situ spectrum acquisition can track the conversion of p-nitrothiophenol to 4, 4‧-dimercapto-azobenzene at ambient temperature. In particular, as a result of chemical catalysis of Ag elements and strong plasmon-molecule coupling, catalytic kinetics of nanoporous Au-Ag shell is 79.2–123.8 times faster than Au nanoparticles (NPs), and 2.2–3.3 times faster than Ag NPs. This investigation offers a route to design superior catalysts to integrate chemical and plasmonic catalysis.

  8. Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy

    PubMed Central

    Ladoux, Benoit; Quivy, Jean-Pierre; Doyle, Patrick; Roure, Olivia du; Almouzni, Geneviève; Viovy, Jean-Louis

    2000-01-01

    Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained. PMID:11114182

  9. PLASMA EFFECTS ON FAST PAIR BEAMS. II. REACTIVE VERSUS KINETIC INSTABILITY OF PARALLEL ELECTROSTATIC WAVES

    SciTech Connect

    Schlickeiser, R.; Krakau, S.; Supsar, M. E-mail: steffen.krakau@rub.de

    2013-11-01

    The interaction of TeV gamma-rays from distant blazars with the extragalactic background light produces relativistic electron-positron pair beams by the photon-photon annihilation process. Using the linear instability analysis in the kinetic limit, which properly accounts for the longitudinal and the small but finite perpendicular momentum spread in the pair momentum distribution function, the growth rate of parallel propagating electrostatic oscillations in the intergalactic medium is calculated. Contrary to the claims of Miniati and Elyiv, we find that neither the longitudinal nor the perpendicular spread in the relativistic pair distribution function significantly affect the electrostatic growth rates. The maximum kinetic growth rate for no perpendicular spread is even about an order of magnitude greater than the corresponding reactive maximum growth rate. The reduction factors in the maximum growth rate due to the finite perpendicular spread in the pair distribution function are tiny and always less than 10{sup –4}. We confirm earlier conclusions by Broderick et al. and our group that the created pair beam distribution function is quickly unstable in the unmagnetized intergalactic medium. Therefore, there is no need to require the existence of small intergalactic magnetic fields to scatter the produced pairs, so that the explanation (made by several authors) for the Fermi non-detection of the inverse Compton scattered GeV gamma-rays by a finite deflecting intergalactic magnetic field is not necessary. In particular, the various derived lower bounds for the intergalactic magnetic fields are invalid due to the pair beam instability argument.

  10. Fast and slow crystal growth kinetics in glass-forming melts.

    PubMed

    Orava, J; Greer, A L

    2014-06-07

    Published values of crystal growth rates are compared for supercooled glass-forming liquids undergoing congruent freezing at a planar crystal-liquid interface. For the purposes of comparison pure metals are considered to be glass-forming systems, using data from molecular-dynamics simulations. For each system, the growth rate has a maximum value U(max) at a temperature T(max) that lies between the glass-transition temperature T(g) and the melting temperature T(m). A classification is suggested, based on the lability (specifically, the propensity for fast crystallization), of the liquid. High-lability systems show "fast" growth characterized by a high U(max), a low T(max)/T(m), and a very broad peak in U vs. T/T(m). In contrast, systems showing "slow" growth have a low U(max), a high T(max)/T(m), and a sharp peak in U vs. T/T(m). Despite the difference of more than 11 orders of magnitude in U(max) seen in pure metals and in silica, the range of glass-forming systems surveyed fit into a common pattern in which the lability increases with lower reduced glass-transition temperature (T(g)/T(m)) and higher fragility of the liquid. A single parameter, a linear combination of T(g)/T(m) and fragility, can show a good correlation with U(max). For all the systems, growth at U(max) is coupled to the atomic/molecular mobility in the liquid. It is found that, across the diversity of glass-forming systems, T(max)/T(g) = 1.48 ± 0.15.

  11. Efficient hydrogen production on MoNi4 electrocatalysts with fast water dissociation kinetics

    NASA Astrophysics Data System (ADS)

    Zhang, Jian; Wang, Tao; Liu, Pan; Liao, Zhongquan; Liu, Shaohua; Zhuang, Xiaodong; Chen, Mingwei; Zschech, Ehrenfried; Feng, Xinliang

    2017-05-01

    Various platinum-free electrocatalysts have been explored for hydrogen evolution reaction in acidic solutions. However, in economical water-alkali electrolysers, sluggish water dissociation kinetics (Volmer step) on platinum-free electrocatalysts results in poor hydrogen-production activities. Here we report a MoNi4 electrocatalyst supported by MoO2 cuboids on nickel foam (MoNi4/MoO2@Ni), which is constructed by controlling the outward diffusion of nickel atoms on annealing precursor NiMoO4 cuboids on nickel foam. Experimental and theoretical results confirm that a rapid Tafel-step-decided hydrogen evolution proceeds on MoNi4 electrocatalyst. As a result, the MoNi4 electrocatalyst exhibits zero onset overpotential, an overpotential of 15 mV at 10 mA cm-2 and a low Tafel slope of 30 mV per decade in 1 M potassium hydroxide electrolyte, which are comparable to the results for platinum and superior to those for state-of-the-art platinum-free electrocatalysts. Benefiting from its scalable preparation and stability, the MoNi4 electrocatalyst is promising for practical water-alkali electrolysers.

  12. BioRef II—Neutron reflectometry with relaxed resolution for fast, kinetic measurements at HZB

    NASA Astrophysics Data System (ADS)

    Trapp, M.; Steitz, R.; Kreuzer, M.; Strobl, M.; Rose, M.; Dahint, R.

    2016-10-01

    We present an upgrade to the time-of-flight neutron reflectometer BioRef at the research reactor BER II of the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). Through the integration of an additional chopper into the existing setup, the available wavelength resolution is significantly extended. Now two distinct operation modes can be used: a high resolution mode with Δλ/λ ranging from 1% to 5%, which allows for the investigation of thick films up to 4000 Å, and a high flux mode with Δλ/λ = 7%-11%. In the high flux mode, reflectivity curves from 0.007 Å-1 to 0.2 Å-1 with three angular settings can be recorded in 7 min. For a single angular setting and its respective window in Q-space, a time resolution of even less than 4 min is reached. The different configurations are documented by respective measurements (a) on a Ni-Ti multilayer and (b) the swelling kinetics of a solid-supported phospholipid coating upon incubation in a polyelectrolyte solution.

  13. Linear and nonlinear thermodynamics of a kinetic heat engine with fast transformations

    NASA Astrophysics Data System (ADS)

    Cerino, Luca; Puglisi, Andrea; Vulpiani, Angelo

    2016-04-01

    We investigate a kinetic heat engine model composed of particles enclosed in a box where one side acts as a thermostat and the opposite side is a piston exerting a given pressure. Pressure and temperature are varied in a cyclical protocol of period τ : their relative excursions, δ and ɛ , respectively, constitute the thermodynamic forces dragging the system out of equilibrium. The analysis of the entropy production of the system allows us to define the conjugated fluxes, which are proportional to the extracted work and the consumed heat. In the limit of small δ and ɛ the fluxes are linear in the forces through a τ -dependent Onsager matrix whose off-diagonal elements satisfy a reciprocal relation. The dynamics of the piston can be approximated, through a coarse-graining procedure, by a Klein-Kramers equation which—in the linear regime—yields analytic expressions for the Onsager coefficients and the entropy production. A study of the efficiency at maximum power shows that the Curzon-Ahlborn formula is always an upper limit which is approached at increasing values of the thermodynamic forces, i.e., outside of the linear regime. In all our analysis the adiabatic limit τ →∞ and the the small-force limit δ ,ɛ →0 are not directly related.

  14. BioRef II-Neutron reflectometry with relaxed resolution for fast, kinetic measurements at HZB.

    PubMed

    Trapp, M; Steitz, R; Kreuzer, M; Strobl, M; Rose, M; Dahint, R

    2016-10-01

    We present an upgrade to the time-of-flight neutron reflectometer BioRef at the research reactor BER II of the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). Through the integration of an additional chopper into the existing setup, the available wavelength resolution is significantly extended. Now two distinct operation modes can be used: a high resolution mode with Δλ/λ ranging from 1% to 5%, which allows for the investigation of thick films up to 4000 Å, and a high flux mode with Δλ/λ = 7%-11%. In the high flux mode, reflectivity curves from 0.007 Å(-1) to 0.2 Å(-1) with three angular settings can be recorded in 7 min. For a single angular setting and its respective window in Q-space, a time resolution of even less than 4 min is reached. The different configurations are documented by respective measurements (a) on a Ni-Ti multilayer and (b) the swelling kinetics of a solid-supported phospholipid coating upon incubation in a polyelectrolyte solution.

  15. Acquisition and analysis of fast single channel kinetic data on an Apple IIe microcomputer.

    PubMed

    Kits, K S; Mos, G J; Leeuwerik, F J; Wattel, C

    1987-05-01

    We describe a method for acquisition and analysis of single channel kinetic data, that uses a ubiquitous microcomputer, the Apple IIe (and is thus cheap), and is yet sensitive and powerful (analysis of up to 6000 events per run of the program; event detection only limited by the amplifier bandwidth). For data acquisition, the original record is converted into an idealized record of open and closed times, using half amplitude threshold analysis or a two-cursor window discriminator. A Time Interval Meter (TIM) has been developed that measures the durations of the TTL-signal from the window discriminator and stores up to 256 intervals in a FIFO-buffer. Each cycle of the TIM is completed within 1 microsecond. The, largely Pascal, software reads the intervals from the FIFO-buffer and stores them in the main memory of the microcomputer (cycle time 70 microseconds). The package is completed with Pascal programs for statistical analysis, including histogram construction of open and closed times, non-linear fit of up to 3 exponentials and correlation analysis.

  16. A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics

    PubMed Central

    Subach, Oksana M.; Entenberg, David; Condeelis, John S.; Verkhusha, Vladislav V.

    2012-01-01

    Fluorescent proteins photoswitchable with non-cytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells. PMID:22900938

  17. Total Kinetic Energy Release in the Fast Neutron Induced Fission of 235U

    NASA Astrophysics Data System (ADS)

    Loveland, Walter; Yanez, Ricardo

    2016-09-01

    We have measured the total kinetic energy (TKE) release, its variance and associated fission product mass distributions for the neutron induced fission of 235U for En = 2-90 MeV using the 2E method. The neutron energies were determined,event by event, by time of flight measurements with the white spectrum neutron beam from LANSCE. The TKE decreases with increasing neutron energy. This TKE decrease is due to increasing symmetric fission (and decreasing asymmetric fission)with increasing neutron energy, in accord with Brosa model predictions. Our measurement of the TKE release for 235U(nth,f) is in excellent agreement with the known value, indicating our measurements are absolute measurements. The TKE variances are sensitive indicators of nth chance fission. Due to the occurrence of nth chance fission and pre-fission neutron emission, the average fissioning system and its excitation energy is a complex function of the incident neutron energy. Detailed comparisons of our data with previous measurements will be made. This work was supported, in part, by the Director, Office of Energy Research, Division of Nuclear Physics of the Office of High Energy and Nuclear Physics of the U.S. Department of Energy under Grant DE-SC0014380.

  18. Linear and nonlinear thermodynamics of a kinetic heat engine with fast transformations.

    PubMed

    Cerino, Luca; Puglisi, Andrea; Vulpiani, Angelo

    2016-04-01

    We investigate a kinetic heat engine model composed of particles enclosed in a box where one side acts as a thermostat and the opposite side is a piston exerting a given pressure. Pressure and temperature are varied in a cyclical protocol of period τ: their relative excursions, δ and ε, respectively, constitute the thermodynamic forces dragging the system out of equilibrium. The analysis of the entropy production of the system allows us to define the conjugated fluxes, which are proportional to the extracted work and the consumed heat. In the limit of small δ and ε the fluxes are linear in the forces through a τ-dependent Onsager matrix whose off-diagonal elements satisfy a reciprocal relation. The dynamics of the piston can be approximated, through a coarse-graining procedure, by a Klein-Kramers equation which-in the linear regime-yields analytic expressions for the Onsager coefficients and the entropy production. A study of the efficiency at maximum power shows that the Curzon-Ahlborn formula is always an upper limit which is approached at increasing values of the thermodynamic forces, i.e., outside of the linear regime. In all our analysis the adiabatic limit τ→∞ and the the small-force limit δ,ε→0 are not directly related.

  19. Spherical polystyrene-supported chitosan thin film of fast kinetics and high capacity for copper removal.

    PubMed

    Jiang, Wei; Chen, Xubin; Pan, Bingcai; Zhang, Quanxing; Teng, Long; Chen, Yufan; Liu, Lu

    2014-07-15

    In order to accelerate the kinetics and improve the utilization of the surface active groups of chitosan (CS) for heavy metal ion removal, sub-micron-sized polystyrene supported chitosan thin-film was synthesized by the electrostatic assembly method. Glutaraldehyde was used as cross-linking agent. Chitosan thin-film was well coated onto the surface of the polystyrene (PS) beads characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). Their adsorption toward Cu(II) ions was investigated as a function of solution pH, degree of cross-linking, equilibrium Cu(II) ions concentration and contact time. The maximum adsorptive capacity of PS-CS was 99.8 mg/g in the adsorption isotherm study. More attractively, the adsorption equilibrium was achieved in 10 min, which showed superior properties among similar adsorbents. Continuous adsorption-desorption cyclic results demonstrated that Cu(II)-loaded PS-CS can be effectively regenerated by a hydrochloric acid solution (HCl), and the regenerated composite beads could be employed for repeated use without significant capacity loss, indicating the good stability of the adsorbents. The XPS analysis confirmed that the adsorption process was due to surface complexes with atoms of chitosan. Generally, PS beads could be employed as a promising host to fabricate efficient composites that originated from chitosan or other bio-sorbents for environmental remediation. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Resolution of the diffusional paradox predicting infinitely fast kinetics on the nanoscale

    NASA Astrophysics Data System (ADS)

    Beke, D. L.; Erdélyi, Z.

    2006-01-01

    In our paper, we offer a natural resolution for a long-standing paradox in diffusion. We show that the growth rate of the diffusion zone (reaction layer) should not go to infinity with decreasing time (as 1/t ), just because the diffusion permeability of the interface is finite. Expression for the changeover thickness X* between the linear and parabolic regimes of the interface shift in phase separating binary A(B) systems is derived in the framework of a deterministic atomistic model for diffusion. X* lies typically between 0.01 and 300nm , depending on the composition dependence of the diffusion coefficient and the phase separation tendency of the alloy. While in ideal binary alloys with composition independent diffusivity, the deviation from the parabolic law practically cannot be observed, in real systems (where the diffusion coefficient can change several orders of magnitude with the composition), measurable deviations are expected as it was experimentally observed very recently in the Ni/Cu and Au/Ni systems. We also offer an atomistic explanation for the phenomenological interface transfer coefficient K . It measures the finite interface permeability (proportional to the jump frequency across the interface) and thus it controls the shift of the interface at short times (diffusion distances). Although it is almost exclusively accepted in the literature that linear growth kinetics are the result of interface reaction control, our results suggest that the linear or nonparabolic growth of a reaction layer on the nanoscale cannot be automatically interpreted by an interface reaction.

  1. Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein-Bound Ligands.

    PubMed

    Krimmer, Stefan G; Cramer, Jonathan; Betz, Michael; Fridh, Veronica; Karlsson, Robert; Heine, Andreas; Klebe, Gerhard

    2016-12-08

    A previously studied congeneric series of thermolysin inhibitors addressing the solvent-accessible S2' pocket with different hydrophobic substituents showed modulations of the surface water layers coating the protein-bound inhibitors. Increasing stabilization of water molecules resulted in an enthalpically more favorable binding signature, overall enhancing affinity. Based on this observation, we optimized the series by designing tailored P2' substituents to improve and further stabilize the surface water network. MD simulations were applied to predict the putative water pattern around the bound ligands. Subsequently, the inhibitors were synthesized and characterized by high-resolution crystallography, microcalorimetry, and surface plasmon resonance. One of the designed inhibitors established the most pronounced water network of all inhibitors tested so far, composed of several fused water polygons, and showed 50-fold affinity enhancement with respect to the original methylated parent ligand. Notably, the inhibitor forming the most perfect water network also showed significantly prolonged residence time compared to the other tested inhibitors.

  2. Effect of the competition of Cu(II) and Ni(II) on the kinetic and thermodynamic stabilities of Cr(III)-organic ligand complexes using competitive ligand exchange (EDTA).

    PubMed

    Cunha, Graziele da Costa; Goveia, Danielle; Romão, Luciane Pimenta Cruz; de Oliveira, Luciana Camargo

    2015-05-01

    The effect of competition of Cu(II) and Ni(II) on the kinetic stability of Cr(III) complexed with natural organic matter (NOM) was characterized using EDTA exchange with single-stage tangential-flow ultrafiltration. For a water sample from Serra de Itabaiana, 3% of spiked Cr(III) was exchanged, while for a sample from the Itapanhaú River, 7, 10, 10, and 21% was exchanged in experiments using Cr(III) alone and in combination with Cu(II), Ni(II), or Cu(II) + Ni(II), respectively. Times required to reach exchange equilibrium with EDTA were less than 360 min. The influence of competition from Ni(II) and Cu(II) on the availability of complexed Cr(III) was low, demonstrating preference of the ligand sites for Cr(III). This was correlated with sample humification, as confirmed by EPR and (13)C NMR analyses. Exchange efficiency was in the order Cu > Ni > Cr, and the process could be readily described by first order kinetics, with average rate constants of 0.35-0.37 h(-1).

  3. Surface Interrogation Scanning Electrochemical Microscopy of Ni(1-x)Fe(x)OOH (0 < x < 0.27) Oxygen Evolving Catalyst: Kinetics of the "fast" Iron Sites.

    PubMed

    Ahn, Hyun S; Bard, Allen J

    2016-01-13

    Nickel-iron mixed metal oxyhydroxides have attracted significant attention as an oxygen evolution reaction (OER) catalyst for solar fuel renewable energy applications. Here, we performed surface-selective and time-dependent redox titrations to directly measure the surface OER kinetics of Ni(IV) and Fe(IV) in NiOOH, FeOOH, and Ni(1-x)Fe(x)OOH (0 < x < 0.27) electrodes. Most importantly, two types of surface sites exhibiting "fast" and "slow" kinetics were found, where the fraction of "fast" sites in Ni(1-x)Fe(x)OOH matched the iron atom content in the film. This finding provides experimental support to the theory-proposed model of active sites in Ni(1-x)Fe(x)OOH. The OER rate constant of the "fast" site was 1.70 s(-1) per atom.

  4. Fast and slow crystal growth kinetics in glass-forming melts

    SciTech Connect

    Orava, J.; Greer, A. L.

    2014-06-07

    Published values of crystal growth rates are compared for supercooled glass-forming liquids undergoing congruent freezing at a planar crystal-liquid interface. For the purposes of comparison pure metals are considered to be glass-forming systems, using data from molecular-dynamics simulations. For each system, the growth rate has a maximum value U{sub max} at a temperature T{sub max} that lies between the glass-transition temperature T{sub g} and the melting temperature T{sub m}. A classification is suggested, based on the lability (specifically, the propensity for fast crystallization), of the liquid. High-lability systems show “fast” growth characterized by a high U{sub max}, a low T{sub max} / T{sub m}, and a very broad peak in U vs. T / T{sub m}. In contrast, systems showing “slow” growth have a low U{sub max}, a high T{sub max} / T{sub m}, and a sharp peak in U vs. T / T{sub m}. Despite the difference of more than 11 orders of magnitude in U{sub max} seen in pure metals and in silica, the range of glass-forming systems surveyed fit into a common pattern in which the lability increases with lower reduced glass-transition temperature (T{sub g} / T{sub m}) and higher fragility of the liquid. A single parameter, a linear combination of T{sub g} / T{sub m} and fragility, can show a good correlation with U{sub max}. For all the systems, growth at U{sub max} is coupled to the atomic/molecular mobility in the liquid. It is found that, across the diversity of glass-forming systems, T{sub max} / T{sub g} = 1.48 ± 0.15.

  5. Effect of pH and organic ligands on the kinetics of smectite dissolution at 25 °C

    NASA Astrophysics Data System (ADS)

    Golubev, Sergey V.; Bauer, Andreas; Pokrovsky, Oleg S.

    2006-09-01

    Forward dissolution rates of Na-Montmorillonite (Wyoming) SWy-2 smectite (Ca 0.06Na 0.56)[Al 3.08Fe(III) 0.38Mg 0.54] [Si 7.93 Al 0.07]O 20(OH) 4 were measured at 25 °C in a mixed-flow reactor equipped with interior dialysis compartment (6-8 kDa membrane) as a function of pH (1-12), dissolved carbonate (0.5-10 mM), phosphate (10 -5 to 0.03 M), and nine organic ligands (acetate, oxalate, citrate, EDTA, alginate, glucuronic acid, 3,4-dihydroxybenzoic acid, gluconate, and glucosamine) in the concentration range from 10 -5 to 0.03 M. In organic-free solutions, the Si-based rates decrease with increasing pH at 1 ⩽ pH ⩽ 8 with a slope close to -0.2. At 9 ⩽ pH ⩽ 12, the Si-based rates increase with a slope of ˜0.3. In contrast, non-stoichiometric Mg release weakly depends on pH at 1 ⩽ pH ⩽ 12 and decreases with increasing pH. The empirical expression describing Si-release rates [ R, mol/cm 2/s] obtained in the present study at 25 °C, I = 0.01 M is given by R=2.2·10-17·aH0.21+1.0·10-20+6·10-17·aOH0.33 At circum-neutral pH, the Si-release-based dissolution is promoted by the addition of the following ligands ranked by decreasing effectiveness: EDTA > 3,4-DHBA > citrate ⩾ oxalate. Phosphate, glucuronate, glucosamine, gluconate, alginate, and acetate act as inhibitors of dissolution and HCO 3-, CO 32- exhibit no effect on dissolution rate. Non-stoichiometric, non-steady-state Mg release was very weakly affected by the presence of ligands. Analysis of reacted solid products using XRD, FT-IR, and XPS revealed no major change in structure, surface chemical composition or specific surface area as a function of pH, ligand concentration, and duration of experiments. Ligand-affected rates re-calculated to constant pH were interpreted using a phenomenological equation which postulates the Langmurian adsorption of a ligand on surface sites. Overall, results of this study demonstrate that very high concentrations (0.001-0.01 M) of organic ligands, whether they are

  6. A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper.

    PubMed

    Bavan, Selvan; Straub, Volko A; Blaxter, Mark L; Ennion, Steven J

    2009-01-20

    vertebrates and invertebrates. Furthermore, several characteristics of HdP2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make HdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.

  7. A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

    PubMed Central

    Bavan, Selvan; Straub, Volko A; Blaxter, Mark L; Ennion, Steven J

    2009-01-01

    vertebrates and invertebrates. Furthermore, several characteristics of HdP2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make HdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms. PMID:19154569

  8. Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation.

    PubMed

    Martinelli, Leonardo Krás Borges; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Breda, Ardala; Selbach, Bruna Pelegrim; Santos, Diógenes Santiago; Basso, Luiz Augusto

    2011-04-01

    Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

  9. Does a fast nuclear magnetic resonance spectroscopy- and X-ray crystallography hybrid approach provide reliable structural information of ligand-protein complexes? A case study of metalloproteinases.

    PubMed

    Isaksson, Johan; Nyström, Susanne; Derbyshire, Dean; Wallberg, Hans; Agback, Tatiana; Kovacs, Helena; Bertini, Ivano; Giachetti, Andrea; Luchinat, Claudio

    2009-03-26

    A human matrix metalloproteinase (MMP) hydroxamic acid inhibitor (CGS27023A) was cross-docked into 15 MMP-12, MMP-13, MMP-9, and MMP-1 cocrystal structures. The aim was to validate a fast protocol for ligand binding conformation elucidation and to probe the feasibility of using inhibitor-protein NMR contacts to dock an inhibitor into related MMP crystal structures. Such an approach avoids full NMR structure elucidation, saving both spectrometer- and analysis time. We report here that for the studied MMPs, one can obtain docking results well within 1 A compared to the corresponding reference X-ray structure, using backbone amide contacts only. From the perspective of the pharmaceutical industry, these results are relevant for the binding studies of inhibitor series to a common target and have the potential advantage of obtaining information on protein-inhibitor complexes that are difficult to crystallize.

  10. Computational studies of the first order kinetic reactions for mononuclear copper(II) complexes having a hard-soft NS donor ligand

    NASA Astrophysics Data System (ADS)

    Zaky, R. R.; Yousef, T. A.; Abdelghany, A. M.

    2014-09-01

    The chelation behaviour of 4-((E)-2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carbonitrile (HL) towards Cu(II) ions has been investigated. These Cu(II) complexes are characterized by elemental analyses, molar-solid conductance, ESR, FTIR and electronic spectral studies. Also, the kinetic and thermodynamic parameters (Ea, A, ΔH, ΔS, ΔG) for all thermal decomposition steps have been evaluated using Coats-Redfern and Horowitz-Metzger methods. Furthermore, antimicrobial activity of the ligand and its complexes were studied against Gram-negative bacteria: Escherichia coli, Gram-positive Bacillus cereus, Bacillus subtilis and pathogenic fungi Pseudomonas aeruginosa by using minimum inhibitory concentrations (MICs) method.

  11. Computational studies of the first order kinetic reactions for mononuclear copper(II) complexes having a hard-soft NS donor ligand.

    PubMed

    Zaky, R R; Yousef, T A; Abdelghany, A M

    2014-09-15

    The chelation behaviour of 4-((E)-2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)-1-(4-methoxyphenyl)-1H-pyrrole-3-carbonitrile (HL) towards Cu(II) ions has been investigated. These Cu(II) complexes are characterized by elemental analyses, molar-solid conductance, ESR, FTIR and electronic spectral studies. Also, the kinetic and thermodynamic parameters (Ea, A, ΔH, ΔS, ΔG) for all thermal decomposition steps have been evaluated using Coats-Redfern and Horowitz-Metzger methods. Furthermore, antimicrobial activity of the ligand and its complexes were studied against Gram-negative bacteria: Escherichia coli, Gram-positive Bacillus cereus, Bacillus subtilis and pathogenic fungi Pseudomonas aeruginosa by using minimum inhibitory concentrations (MICs) method.

  12. Equivalence of on-Lattice Stochastic Chemical Kinetics with the Well-Mixed Chemical Master Equation in the Limit of Fast Diffusion.

    PubMed

    Stamatakis, Michail; Vlachos, Dionisios G

    2011-12-14

    Well-mixed and lattice-based descriptions of stochastic chemical kinetics have been extensively used in the literature. Realizations of the corresponding stochastic processes are obtained by the Gillespie stochastic simulation algorithm and lattice kinetic Monte Carlo algorithms, respectively. However, the two frameworks have remained disconnected. We show the equivalence of these frameworks whereby the stochastic lattice kinetics reduces to effective well-mixed kinetics in the limit of fast diffusion. In the latter, the lattice structure appears implicitly, as the lumped rate of bimolecular reactions depends on the number of neighbors of a site on the lattice. Moreover, we propose a mapping between the stochastic propensities and the deterministic rates of the well-mixed vessel and lattice dynamics that illustrates the hierarchy of models and the key parameters that enable model reduction.

  13. Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques.

    PubMed

    Carver, T E; Rohlfs, R J; Olson, J S; Gibson, Q H; Blackmore, R S; Springer, B A; Sligar, S G

    1990-11-15

    Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of

  14. A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

    PubMed

    O'Flaherty, M; O'Carra, P; McMahon, M; Mulcahy, P

    1999-08-01

    The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).

  15. A fast, open source implementation of adaptive biasing potentials uncovers a ligand design strategy for the chromatin regulator BRD4.

    PubMed

    Dickson, Bradley M; de Waal, Parker W; Ramjan, Zachary H; Xu, H Eric; Rothbart, Scott B

    2016-10-21

    In this communication we introduce an efficient implementation of adaptive biasing that greatly improves the speed of free energy computation in molecular dynamics simulations. We investigated the use of accelerated simulations to inform on compound design using a recently reported and clinically relevant inhibitor of the chromatin regulator BRD4 (bromodomain-containing protein 4). Benchmarking on our local compute cluster, our implementation achieves up to 2.5 times more force calls per day than plumed2. Results of five 1 μs-long simulations are presented, which reveal a conformational switch in the BRD4 inhibitor between a binding competent and incompetent state. Stabilization of the switch led to a -3 kcal/mol improvement of absolute binding free energy. These studies suggest an unexplored ligand design principle and offer new actionable hypotheses for medicinal chemistry efforts against this druggable epigenetic target class.

  16. "Adapted Linear Interaction Energy": A Structure-Based LIE Parametrization for Fast Prediction of Protein-Ligand Affinities.

    PubMed

    Linder, Mats; Ranganathan, Anirudh; Brinck, Tore

    2013-02-12

    We present a structure-based parametrization of the Linear Interaction Energy (LIE) method and show that it allows for the prediction of absolute protein-ligand binding energies. We call the new model "Adapted" LIE (ALIE) because the α and β coefficients are defined by system-dependent descriptors and do therefore not require any empirical γ term. The best formulation attains a mean average deviation of 1.8 kcal/mol for a diverse test set and depends on only one fitted parameter. It is robust with respect to additional fitting and cross-validation. We compare this new approach with standard LIE by Åqvist and co-workers and the LIE + γSASA model (initially suggested by Jorgensen and co-workers) against in-house and external data sets and discuss their applicabilities.

  17. A fast, open source implementation of adaptive biasing potentials uncovers a ligand design strategy for the chromatin regulator BRD4

    NASA Astrophysics Data System (ADS)

    Dickson, Bradley M.; de Waal, Parker W.; Ramjan, Zachary H.; Xu, H. Eric; Rothbart, Scott B.

    2016-10-01

    In this communication we introduce an efficient implementation of adaptive biasing that greatly improves the speed of free energy computation in molecular dynamics simulations. We investigated the use of accelerated simulations to inform on compound design using a recently reported and clinically relevant inhibitor of the chromatin regulator BRD4 (bromodomain-containing protein 4). Benchmarking on our local compute cluster, our implementation achieves up to 2.5 times more force calls per day than plumed2. Results of five 1 μs-long simulations are presented, which reveal a conformational switch in the BRD4 inhibitor between a binding competent and incompetent state. Stabilization of the switch led to a -3 kcal/mol improvement of absolute binding free energy. These studies suggest an unexplored ligand design principle and offer new actionable hypotheses for medicinal chemistry efforts against this druggable epigenetic target class.

  18. Equilibrium, Kinetic and Structural Properties of Gallium(III) and Some Divalent Metal Complexes Formed with the New DATA(m) and DATA(5m) Ligands.

    PubMed

    Farkas, Edit; Nagel, Johannes; Waldron, Bradley P; Parker, David; Tóth, Imre; Brücher, Ernő; Rösch, Frank; Baranyai, Zsolt

    2017-08-01

    The development of (68) Ge/(68) Ga generators has made the positron-emitting (68) Ga isotope widely accessible and raised interest in new chelate complexes of Ga(3+) . The hexadentate 1,4-di(acetate)-6-methyl[amino(methyl)acetate]perhydro-1,4-diazepane (DATA(m) ) ligand and its bifunctional analogue, 1,4-di(acetate)-6-pentanoic acid[amino(methyl)acetate]perhydro-1,4-diazepane (DATA(5m) ), rapidly form complexes with (68) Ga in high radiochemical yield. The stability constants of DATA(m) and DATA(5m) complexes formed with Ga(3+) , Zn(2+) , Cu(2+) , Mn(2+) and Ca(2+) have been determined by using pH potentiometry, spectrophotometry (Cu(2+) ) and (1) H and (71) Ga NMR spectroscopy (Ga(3+) ). The stability constants of Ga(DATA(m) ) and Ga(DATA(5m) ) complexes are slightly higher than those of Ga(AAZTA). The species distribution calculations indicated the predominance of Ga(L)OH mixed-hydroxo complexes at physiological pH. The (1) H and (71) Ga NMR spectroscopy studies provided information about the coordinated functional groups of ligands and on the kinetics of exchange between the Ga(L) and Ga(L)OH complexes. The transmetalation reactions between the Ga(L) complexes and Cu(2+) citrate (6ligand-exchange reactions between the Ga(L)OH complexes and transferrin. The equilibrium and kinetic data indicate that the Ga(DATA(5m) ) complex is a good (68) Ga-based radiodiagnostic candidate. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A kinetic, mechanistic, and molecular mechanics investigation of olefin insertion into organoactinide-hydride bonds. Metal, olefin, ancillary ligand, and diastereoselection effects

    SciTech Connect

    Lin, Zerong; Marks, T.J. )

    1990-07-04

    This contribution reports a kinetic/mechanistic/stereochemical/molecular mechanics study of olefin insertion into the actinide-hydrogen bonds of Cp{prime}{sub 2}An(H)(OR) complexes (Cp{prime} = {eta}{sup 5}-(CH{sub 3}){sub 5}C{sub 5}; An = Th, U; R = achiral or chiral alkyl group). For the reaction Cp{prime}{sub 2}An(H)(O-t-Bu) + cyclohexene (An = Th), the rate law is first order in organoactinide and first order in olefin. For Cp{prime}{sub 2}Th(H)(OCH(t-Bu){sub 2}) + 1-hexene, k{sub Th{minus}H}/k{sub Th{minus}D} = 1.3 (2). A molecular mechanics/ molecular graphics analysis suggests that the sterically most favorable direction of olefin approach toward the actinide center is between the U-H and U-O bonds rather than from the side. These results provide additional insight into ancillary ligand effects on the kinetics of organo-f-element-catalyzed olefin hydrogenation.

  20. Basal and fasting/refeeding-regulated tissue levels of endogenous PPAR-alpha ligands in Zucker rats.

    PubMed

    Izzo, Angelo A; Piscitelli, Fabiana; Capasso, Raffaele; Marini, Pietro; Cristino, Luigia; Petrosino, Stefania; Di Marzo, Vincenzo

    2010-01-01

    N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA) are endogenous lipids that activate peroxisome proliferator-activated receptor-alpha with high and intermediate potency, and exert anorectic and anti-inflammatory actions in rats, respectively. We investigated OEA and PEA tissue level regulation by the nutritional status in lean and obese rats. OEA and PEA levels in the brainstem, duodenum, liver, pancreas, and visceral (VAT) or subcutaneous (SAT) adipose tissues of 7-week-old wild-type (WT) and Zucker rats, fed ad libitum or following overnight food deprivation, with and without refeeding, were measured by liquid chromatography-mass spectrometry. In WT rats, duodenal OEA, but not PEA, levels were reduced by food deprivation and restored by refeeding, whereas the opposite was observed for OEA in the pancreas, and for both mediators in the liver and SAT. In ad lib fed Zucker rats, PEA and OEA levels were up to tenfold higher in the duodenum, slightly higher in the brainstem, and lower in the other tissues. Fasting/refeeding-induced changes in OEA levels were maintained in the duodenum, liver, and SAT, and lost in the pancreas, whereas fasting upregulated this compound also in the VAT. The observed changes in OEA levels in WT rats are relevant to the actions of this mediator on satiety, hepatic and adipocyte metabolism, and insulin release. OEA dysregulation in Zucker rats might counteract hyperphagia in the duodenum, but contribute to hyperinsulinemia in the pancreas, and to fat accumulation in adipose tissues and liver. Changes in PEA levels might be relevant to the inflammatory state of Zucker rats.

  1. Amplification of a fast wave by extracting both the kinetic energy and electrostatic potential energy of a large-orbit relativistic electron beam in a coaxial electrostatic wiggler

    SciTech Connect

    Zhang Shichang

    2010-05-15

    Nonlinear model and simulation technique of the interaction and energy transfer between a fast wave and a large-orbit relativistic electron beam in a coaxial electrostatic wiggler are presented. Unlike the situations in a magnetostatic-wiggler free-electron laser (MWFEL) and in an electron cyclotron maser (ECM), the electrostatic potential of the electrons plays an important role and participates in the energy exchange between the wave and the electron beam. Compared to MWFEL and ECM, the coaxial electrostatic-wiggler configuration has a distinguishing peculiarity that besides the electron-beam's kinetic energy, its electrostatic potential energy can be effectively transferred to the fast wave. Simulation shows that wave could be amplified with ultrahigh gain by extracting both the kinetic energy and electrostatic potential energy of the electron beam.

  2. How Fast Is Fast?

    ERIC Educational Resources Information Center

    Korn, Abe

    1994-01-01

    Presents an activity that enables students to answer for themselves the question of how fast a body must travel before the nonrelativistic expression must be replaced with the correct relativistic expression by deciding on the accuracy required in describing the kinetic energy of a body. (ZWH)

  3. Dissolution Kinetics of Arsenopyrite in the Presence of Iron(III)-Sequestering Biogenic Ligands at pH 5.

    NASA Astrophysics Data System (ADS)

    Cornejo-Garrido, H.; Fernandez-Lomelin, P.; Guzman-Mendoza, J.; Sedov, S.; Cervini-Silva, J.

    2007-05-01

    Arsenopyrite is one of the most important natural sources of Arsenic on Earth. Arsenopyrite is relatively insoluble in pure water. That is not the case if it is exposed to environmental conditions. Notably, arsenopyrite surfaces exposed to biological activity undergo changes in lattice energy, surface morphology, particle size, and other properties, typical of mineral dissolution. Iron biogeochemical cycling is pivotal to electron transferring in nature. Therein, the need to further scrutinize on the mechanism of arsenopyrite dissolution induced by biological activity. In oxic environments, such as highly weathered soils or surficial seawater, microorganisms and higher plants produce biogenic ligands such as siderophores to mobilize Fe that otherwise would be unavailable. Siderophores ligands facilitate the dissolution of natural particles that represent a primary reservoir of iron. In this paper we study the stability of arsenopyrite in the presence of desferrioxamine (DFO-B), a common siderophore ligand, at pH 5. Arsenopyrite specimens from mines from Panasqueira, Portugal, were used for this study. Batch dissolution experiments of arsenopyrite (1 g L-1) in the presence of DFO-B ([DFO-B]0 - 200 ?M) were conducted for 7 days. The initial pH was adjusted to 5. Samples were stirred at 150 rpm. Sieving was conducted to homogeneized the particle size 0.149-0.1mm before conducting the dissolution experiments. Corresponding experiments in the absence of DFO-B for the purpose of comparison were also conducted. Analyses for soluble metals were conducted by AA and ICP-AES. Surface characterization was conducted by XRD and SEM-EDX. Incrustations of Pb(0) were detected in the arsenopyrite samples used for this study. In the presence of DFO-B, releases of Fe, As, and Pb showed positive trends with time. A shallower dependency was observed for release of Fe, As, and Pb in the presence of water only under similar experimental conditions. Detected concentrations of Fe, As, and

  4. Kinetic Models of Fast Solar Wind Driven by Imbalanced Ion Cyclotron Dissipation - What Will Solar Probe See?

    NASA Astrophysics Data System (ADS)

    Isenberg, P. A.; Vasquez, B. J.

    2012-12-01

    In previous work (e.g. Isenberg & Vasquez, ApJ, 731, 88, 2011), we have shown that resonant dissipation of a turbulently maintained power-law spectrum of ion cyclotron waves can produce a reasonable fast solar wind flow. Kinetic modeling of this ion heating in the expanding collisionless coronal hole must also take into account the effects of gravity, charge-separation electric field, mirror force, inertial force in the accelerating plasma, and ponderomotive Alfvén wave pressure. The combined action of all these processes leads to a characteristic evolution of the proton distribution function, some aspects of which may be independent of the actual mechanism for the perpendicular heating. Our previous model used resonant wave intensities that were "balanced" in that we took the power in outward-propagating waves and sunward-propagating waves to be equal. We also were limited by computational considerations to heliocentric radial positions < 6 Rs. Here, we consider imbalanced cases where the outward-propagating intensities of resonant waves are larger than the sunward intensities, as would be expected from reflection models of turbulent evolution in the solar atmosphere. We also extend our computations to beyond the Alfvenic critical point, into the radial range to be explored by Solar Probe Plus. We will present model results for the solar wind speeds and temperatures as functions of heliocentric radius for various ratios of sunward-to-outward wave intensities. We will also show detailed shapes of the proton distribution function and discuss their radial evolution with particular emphasis on the region accessible to the planned Solar Probe Plus mission.

  5. A macrocyclic ligand able to bind gallium(III) by preorganized pendant arms; coordination and kinetic studies.

    PubMed

    Ambrosi, Gianluca; Boggioni, Alessia; Formica, Mauro; Fusi, Vieri; Giorgi, Luca; Lucarini, Simone; Micheloni, Mauro; Secco, Fernando; Venturini, Marcella; Zappia, Giovanni

    2005-02-07

    The equilibria and kinetics of the binding of gallium(III) to 4-(N),10-(N)-bis[2-(3-hydroxo-2-oxo-2-H-pyridine-1-y1)acetamido]-1,7-dimethyl-1,4,7,10-tetraazacyclododecane (L) were investigated in acidic medium at ionic strength 1 M (NaClO4). Spectrophotometric titrations in the UV region revealed that L is able to bind Ga3+ also at high H+ concentration. The kinetic (stopped-flow) experiments are interpreted on the basis of three parallel reaction paths (i) M3+ + H2L2+ = M(H2L)5+ where M(H2L)5+ is in a steady state, (ii) M(OH)2+ + H2L2+ = M(HL)4+ + H2O and (iii) M(OH)2+ + HL+ = ML3+ + H2O. The first-order rate constants for conversion of the outer-sphere into the inner-sphere complexes are similar to those of the Ga(III)/tropolone system which is known to react according to the dissociative Id mechanism and to the relevant rate constants for water exchange at the metal ion. The effects of pH on the UV-Vis absorption, fluorescence emission properties and NMR spectral features on the Ga(III)/L system were also investigated. Spectrophotometric titrations in the UV region reveal that, in acid medium the prevailing species is M(HL)4+ whereas the chelate ML3+ prevails for [H+] < 0.01 M. The results indicate metal coordination at the oxygen atoms of the 3-hydroxo-2-oxopyridine residues.

  6. CO and O2 binding to pseudo-tetradentate ligand-copper(I) complexes with a variable N-donor moiety: kinetic/thermodynamic investigation reveals ligand-induced changes in reaction mechanism.

    PubMed

    Lucas, Heather R; Meyer, Gerald J; Karlin, Kenneth D

    2010-09-22

    The kinetics, thermodynamics, and coordination dynamics are reported for O(2) and CO 1:1 binding to a series of pseudo-tetradentate ligand-copper(I) complexes ((D)LCu(I)) to give Cu(I)/O(2) and Cu(I)/CO product species. Members of the (D)LCu(I) series possess an identical tridentate core structure where the cuprous ion binds to the bispicolylamine (L) fragment. (D)L also contains a fourth variable N-donor moiety {D = benzyl (Bz); pyridyl (Py); imidazolyl (Im); dimethylamino (NMe(2)); (tert-butylphenyl)pyridyl (TBP); quinolyl (Q)}. The structural characteristics of (D)LCu(I)-CO and (D)LCu(I) are detailed, with X-ray crystal structures reported for (TBP)LCu(I)-CO, (Bz)LCu(I)-CO, and (Q)LCu(I). Infrared studies (solution and solid-state) confirm that (D)LCu(I)-CO possess the same four-coordinate core structure in solution with the variable D moiety "dangling", i.e., not coordinated to the copper(I) ion. Other trends observed for the present series appear to derive from the degree to which the D-group interacts with the cuprous ion center. Electrochemical studies reveal close similarities of behavior for (Im)LCu(I) and (NMe(2))LCu(I) (as well as for (TBP)LCu(I) and (Q)LCu(I)), which relate to the O(2) binding kinetics and thermodynamics. Equilibrium CO binding data (K(CO), ΔH°, ΔS°) were obtained by conducting UV-visible spectrophotometric CO titrations, while CO binding kinetics and thermodynamics (k(CO), ΔH(double dagger), ΔS(double dagger)) were measured through variable-temperature (193-293 K) transient absorbance laser flash photolysis experiments, λ(ex) = 355 nm. Carbon monoxide dissociation rate constants (k(-CO)) and corresponding activation parameters (ΔH(double dagger), ΔS(double dagger)) have also been obtained. CO binding to (D)LCu(I) follows an associative mechanism, with the increased donation from D leading to higher k(CO) values. Unlike observations from previous work, the K(CO) values increased as the k(CO) and k(-CO) values declined; the

  7. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  8. Kinetic studies of the binding of acridinecarboxamide topoisomerase poisons to DNA: implications for mode of binding of ligands with uncharged chromophores.

    PubMed

    Wakelin, Laurence P G; Adams, Adrienne; Denny, William A

    2002-02-14

    We have used stopped-flow spectrophotometry and the sodium dodecyl sulfate sequestration technique to study the kinetics of dissociation of DNA complexes of the mixed topoisomerase I/II poison N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (termed DACA) and a range of related linear tricyclic carboxamides with neutral chromophores. Complexes of DACA and related acridine and phenazinecarboxamides bearing an N,N-dimethylaminoethyl side chain dissociate from calf thymus DNA by a kinetic pathway involving four discernible steps in a manner similar to complexes of N-[(2-dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (termed 9-amino-DACA). We infer from these findings that the side chains of DACA, its phenazine homologue, and 9-amino-DACA make comparable interactions with the DNA base pairs. In the case of 9-amino-DACA, a selective topoisomerase II poison, these are known, by crystallographic analysis, to involve hydrogen-bonding interactions between the protonated dimethylammonium group of the side chain and the O6/N7 atoms of guanine and to include a bridging water molecule hydrogen bonded to the carboxamide group and a phosphate oxygen. By contrast, we find that other linear tricyclic carboxamides with neutral chromophores which lack a peri nitrogen atom and are biologically inactive dissociate from DNA by a different mechanism in which it appears their side chains fail to interact with guanine. We conclude that the ability of the carboxamide group to lie preferentially in the plane of the chromophore, so facilitating the dimethylammonium-guanine hydrogen bond and ensuring maintenance of the water-bridged carboxamide-phosphate interaction, is a critical requirement for antitumor activity among ligands of the linear tricyclic carboxamide class. However, unlike the situation for 9-amino-DACA, for ligands with uncharged chromophores containing peri nitrogen atoms such as DACA, this outcome is possible with the 4-carboxamide group rotated cis or trans with respect

  9. Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes

    PubMed Central

    Rajapaksha, Harinda; Forbes, Briony E.

    2015-01-01

    The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His4, Tyr15, Thr49, Ile51) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I. PMID:26217307

  10. The SH2 domains of inositol polyphosphate 5-phosphatases SHIP1 and SHIP2 have similar ligand specificity but different binding kinetics.

    PubMed

    Zhang, Yanyan; Wavreille, Anne-Sophie; Kunys, Andrew R; Pei, Dehua

    2009-11-24

    SH2 domain-containing inositol 5-phosphatases 1 (SHIP1) and 2 (SHIP2) are structurally similar proteins that catalyze the degradation of lipid secondary messenger phosphatidylinositol 3,4,5-triphosphate to produce phosphatidylinositol 3,4-diphosphate. Despite their high sequence identity (51%), SHIP1 and SHIP2 share little overlap in their in vivo functions. In this work, the sequence specificity of the SHIP2 SH2 domain was systematically defined through the screening of a combinatorial pY peptide library. Comparison of its specificity profile with that of the SHIP1 SH2 domain showed that the two SH2 domains have similar specificities, both recognizing pY peptides of the consensus sequence pY[S/Y][L/Y/M][L/M/I/V], although there are also subtle differences such as the tolerance of an arginine at the pY + 1 position by the SHIP2 but not SHIP1 SH2 domain. Surface plasmon resonance analysis of their interaction with various pY peptides suggested that the two domains have similar binding affinities but dramatically different binding kinetics, with the SHIP1 SH2 domain having fast association and dissociation rates while the SHIP2 domain showing apparent slow-binding behavior. Site-directed mutagenesis and kinetic studies indicated that the SHIP2 SH2 domain exists as a mixture of two conformational isomers. The major, inactive isomer apparently contains two cis peptidyl-prolyl bonds at positions 88 and 105, whereas the minor, active isomer has both proline residues in their trans configuration. Cis-trans isomerization of the peptidyl-prolyl bonds may provide a potential mechanism for regulating the interaction between SHIP2 and pY proteins. These data suggest that a combination of tissue distribution, specificity, and kinetic differences is likely responsible for their in vivo functional differences.

  11. Constructing Kinetic Network Models to Elucidate Mechanisms of Functional Conformational Changes of Enzymes and Their Recognition with Ligands.

    PubMed

    Zhang, L; Jiang, H; Sheong, F K; Pardo-Avila, F; Cheung, P P-H; Huang, X

    2016-01-01

    Enzymes are biological macromolecules that catalyze complex reactions in life. In order to perform their functions effectively and efficiently, enzymes undergo conformational changes between different functional states. Therefore, elucidating the dynamics between these states is essential to understand the molecular mechanisms of enzymes. Although experimental methods such as X-ray crystallography and cryoelectron microscopy can produce high-resolution structures, the detailed conformational dynamics of many enzymes still remain obscure. While molecular dynamics (MD) simulations are able to complement the experiments by providing structure-based dynamics at atomic resolution, it is usually difficult for them to reach the biologically relevant timescales (hundreds of microseconds or longer). Kinetic network models (KNMs), in particular Markov state models (MSMs), hold great promise to overcome this challenge because they can bridge the timescale gap between MD simulations and experimental observations. In this chapter, we review the procedure of constructing KNMs to elucidate the molecular mechanisms of enzymes. First, we will give a general introduction of MSMs, including the methods to construct and validate MSMs. Second, we will present the applications of KNMs to study two important enzymes: the human Argonaute protein and the RNA polymerase II. We conclude by discussing the future perspectives regarding the potential of KNMs to investigate the dynamics of enzymes' functional conformational changes.

  12. Kinetic and ligand binding evidence for two heme A-based terminal oxidases in plasma membranes from Bacillus subtilis.

    PubMed

    Hill, B C; Vo, L; Albanese, J

    1993-02-15

    Detergent-solubilized plasma membranes from Bacillus subtilis have been characterized for their cytochrome oxidase content. Triton X-100-solubilized membranes show high O2 turnover with ascorbate plus TMPD. Reduced-oxidized difference spectroscopy of ascorbate-TMPD-reduced membranes reveals the presence of cytochrome c and cytochrome a. An additional, b-type cytochrome appears when the membranes are reduced with dithionite. Time-resolved difference spectra taken during reduction by ascorbate-TMPD reveal two kinetic forms of heme A-containing cytochromes. There is a high-turnover form that is rapidly reduced upon anaerobiosis, and a second type which is only slowly reduced upon anaerobiosis. The slowly reduced oxidase is distinguished by an alpha-band blue-shifted to 600 nm relative to the 603-nm position observed for high-turnover oxidase. Addition of CO to ascorbate-TMPD-reduced membranes gives a spectrum typical of ferrocytochrome a3-CO, and the intensity corresponds to the total ferrocytochrome a3 concentration. Photolysis of ascorbate-TMPD-reduced, CO-bound membranes indicates that both species are photosensitive with similar rates of recombination. Addition of CO to dithionite-reduced membranes shows an additional CO reactive center that has a spectrum characteristic of cytochrome o. Cyanide blocks complete reduction of high-turnover oxidase by ascorbate plus TMPD, but does not appear to effect slowly reduced oxidase. These results indicate the presence of two different types of cytochrome aa3 oxidase in plasma membranes of B. subtilis.

  13. N-Substituted Benztropine Analogs: Selective Dopamine Transporter Ligands with a Fast Onset of Action and Minimal Cocaine-Like Behavioral Effects

    PubMed Central

    Li, Su-Min; Kopajtic, Theresa A.; O'Callaghan, Matthew J.; Agoston, Gregory E.; Cao, Jianjing; Newman, Amy Hauck

    2011-01-01

    Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted benztropine analogs with a relatively fast onset of effects were studied to assess whether a fast onset of effects would render the effects more similar to those of cocaine. Only one of the compounds increased locomotor activity, and the increases were modest compared with those of 10 to 20 mg/kg cocaine. In rats trained to discriminate 10 mg/kg cocaine from saline none of the compounds produced more than 40% cocaine-like responds up to 2 h after injection. None of the compounds produced place-conditioning when examined up to 90 min after injection, indicating minimal abuse liability. The compounds had 5.6 to 30 nM affinities at the dopamine transporter (DAT), with uniformly lower affinities at norepinephrine and serotonin transporters (from 490-4600 and 1420–7350 nM, respectively). Affinities at muscarinic M1 receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H1 sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-hydroxytryptamine1 (5-HT1), and 5-HT2 receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds. PMID:21088247

  14. Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: Evidence for low-affinity sites and for the involvement of G proteins

    SciTech Connect

    Dunn, S.M.J.; Bladen, C. )

    1991-06-11

    Detailed kinetic studies of the binding of the calcium channel antagonist (+)-({sup 3}H)PN200-110 to membrane preparations form rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-({sup 3}H)PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 {mu}M for the agonist ({plus minus})-Bay K8644 and for the antagonists nifedipine, ({plus minus})-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since ({minus})-PN200-110 (1-200 {mu}M) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) and guanosine 5'-O-(2-thiodiphosphate) (GDP{beta}S) on binding parameters. GTP{gamma}S did increase the ability of ({plus minus})-({sup 3}H)PN200-110. These results suggest that skeletal muscle dihydropyridine receptors have low-affinity binding sites that may be involved in the regulation of calcium channel function and that activation of a guanine nucleotide binding protein may modulate the binding of agonists but not of antagonists to these sites.

  15. Evidence of Kinetic Control of Ligand Binding and Staged Product Release in MurA (enolpyruvyl UDP-GlcNAc synthase)-catalyzed Reactions

    SciTech Connect

    Jackson, S.; Zhang, F; Chindemi, P; Junop, M; Berti, P

    2009-01-01

    MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k{sub cat}/K{sub M} of 520 M{sup -1} s{sup -1}, was far slower than the normal reaction, and 3 x 10{sup 5}-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new 'staged' MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA {center_dot} ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA {center_dot} UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

  16. Evidence of kinetic control of ligand binding and staged product release in MurA (enolpyruvyl UDP-GlcNAc synthase)-catalyzed reactions .

    PubMed

    Jackson, Sean G; Zhang, Fuzhong; Chindemi, Paul; Junop, Murray S; Berti, Paul J

    2009-12-15

    MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

  17. Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions - identification of a new nonpeptide inhibitor using Biacore™ T200.

    PubMed

    Wear, Martin A; Nowicki, Matthew W; Blackburn, Elizabeth A; McNae, Iain W; Walkinshaw, Malcolm D

    2017-04-01

    We have established a refined methodology for generating surface plasmon resonance sensor surfaces of recombinant his-tagged human cyclophilin-A. Our orientation-specific stabilisation approach captures his-tagged protein under 'physiological conditions' (150 mm NaCl, pH 7.5) and covalently stabilises it on Ni(2+)-nitrilotriacetic acid surfaces, very briefly activated for primary amine-coupling reactions, producing very stable and active surfaces (≥ 95% specific activity) of cyclophilin-A. Variation in protein concentration with the same contact time allows straightforward generation of variable density surfaces, with essentially no loss of activity, making the protocol easily adaptable for studying numerous interactions; from very small fragments, ~ 100 Da, to large protein ligands. This new method results in an increased stability and activity of the immobilised protein and allowed us to expand the thermo-kinetic analysis space, and to determine accurate and robust thermodynamic parameters for the cyclophilin-A-cyclosporin-A interaction. Furthermore, the increased sensitivity of the surface allowed identification of a new nonpeptide inhibitor of cyclophilin-A, from a screen of a fragment library. This fragment, 2,3-diaminopyridine, bound specifically with a mean affinity of 248 ± 60 μm. The X-ray structure of this 109-Da fragment bound in the active site of cyclophilin-A was solved to a resolution of 1.25 Å (PDB: 5LUD), providing new insight into the molecular details for a potential new series of nonpeptide cyclophilin-A inhibitors.

  18. Surface Proton Hopping and Fast-Kinetics Pathway of Water Oxidation on Co3O4 (001) Surface

    SciTech Connect

    Pham, Hieu H.; Cheng, Mu-Jeng; Frei, Heinz; Wang, Lin-Wang

    2016-07-18

    A mechanism of water splitting on cobalt oxide surface is proposed, with atomistic thermodynamic and kinetic details. The density-functional theory studies suggest that the oxidation process could proceed with several nonelectrochemical (spontaneous) intermediate steps, following the initial electrochemical hydroxyl-to-oxo conversion. More specifically, the single oxo sites CoIV=O can hop (via surface proton/electron hopping) to form oxo pair CoIV(=O)-O-CoIV=O, which will undergo nucleophilic attack by a water molecule and form the hydroperoxide CoIII -OOH. Encounter with another oxo would generate a superoxo CoIII-OO, followed by the O2 release. Finally the addition and deprotonation of a fresh water molecule will restart the catalytic cycle by forming the hydroxyl CoIII-OH at this active site. Our theoretical investigations indicate that all nonelectrochemical reactions are kinetically fast and thermodynamically downhill. This hypothesis is supported by recent in situ spectroscopic observations of surface superoxo that is stabilized by hydrogen bonding to adjacent hydroxyl group as an intermediate on fast-kinetics Co catalytic site.

  19. Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range.

    PubMed

    Catoire, Laurent J; Damian, Marjorie; Baaden, Marc; Guittet, Eric; Banères, Jean-Louis

    2011-07-01

    The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the (1)H-(1)H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.

  20. The V499G/Y501H mutation impairs fast motor kinetics of prestin and has significance for defining functional independence of individual prestin subunits.

    PubMed

    Homma, Kazuaki; Duan, Chongwen; Zheng, Jing; Cheatham, Mary Ann; Dallos, Peter

    2013-01-25

    Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of prestin provides vital information for unraveling the energy transduction mechanism of prestin. We show that the V499G/Y501H mutation does not deprive prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H prestin forms heteromers with wild-type prestin and that the fast motor kinetics of wild-type prestin is not affected by heteromer formation with V499G/Y501H prestin. These results suggest that prestin subunits are individually functional within a given multimer.

  1. Unchanged content of oxidative enzymes in fast-twitch muscle fibers and V˙O2 kinetics after intensified training in trained cyclists.

    PubMed

    Christensen, Peter M; Gunnarsson, Thomas P; Thomassen, Martin; Wilkerson, Daryl P; Nielsen, Jens Jung; Bangsbo, Jens

    2015-07-01

    The present study examined if high intensity training (HIT) could increase the expression of oxidative enzymes in fast-twitch muscle fibers causing a faster oxygen uptake (V˙O2) response during intense (INT), but not moderate (MOD), exercise and reduce the V˙O2 slow component and muscle metabolic perturbation during INT. Pulmonary V˙O2 kinetics was determined in eight trained male cyclists (V˙O2-max: 59 ± 4 (means ± SD) mL min(-1) kg(-1)) during MOD (205 ± 12 W ~65% V˙O2-max) and INT (286 ± 17 W ~85% V˙O2-max) exercise before and after a 7-week HIT period (30-sec sprints and 4-min intervals) with a 50% reduction in volume. Both before and after HIT the content in fast-twitch fibers of CS (P < 0.05) and COX-4 (P < 0.01) was lower, whereas PFK was higher (P < 0.001) than in slow-twitch fibers. Content of CS, COX-4, and PFK in homogenate and fast-twitch fibers was unchanged with HIT. Maximal activity (μmol g DW(-1) min(-1)) of CS (56 ± 8 post-HIT vs. 59 ± 10 pre-HIT), HAD (27 ± 6 vs. 29 ± 3) and PFK (340 ± 69 vs. 318 ± 105) and the capillary to fiber ratio (2.30 ± 0.16 vs. 2.38 ± 0.20) was unaltered following HIT. V˙O2 kinetics was unchanged with HIT and the speed of the primary response did not differ between MOD and INT. Muscle creatine phosphate was lower (42 ± 15 vs. 66 ± 17 mmol kg DW(-1)) and muscle lactate was higher (40 ± 18 vs. 14 ± 5 mmol kg DW(-1)) at 6 min of INT (P < 0.05) after compared to before HIT. A period of intensified training with a volume reduction did not increase the content of oxidative enzymes in fast-twitch fibers, and did not change V˙O2 kinetics.

  2. Fast-scan cyclic voltammetry reveals that L-Dopa produces regionally selective, bimodal influences on striatal dopamine kinetics in vivo

    PubMed Central

    Harun, R; Munoz, M; Grassi, C; Hare, K; Brough, E; Torres, GE; Grace, AA; Wagner, AK

    2016-01-01

    Parkinson’s disease (PD) is a debilitating condition that is caused by a relatively specific degeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta. Levodopa (L-Dopa) was introduced as a viable treatment option for PD over 40 years ago and still remains the most common and effective therapy for PD. Though the effects of L-Dopa to augment striatal DA production are well known, little is actually known about how L-Dopa alters the kinetics of DA neurotransmission that contribute to its beneficial and adverse effects. In this study, we examined the effects of L-Dopa administration (100mg/kg carbidopa/250mg/kg L-Dopa) on regional electrically stimulated DA response kinetics using fast-scan cyclic voltammetry (FSCV) in anesthetized rats. We demonstrate that L-Dopa enhances DA release in both the dorsal striatum (D-STR) and nucleus accumbens (NAc), but surprisingly causes a delayed inhibition of release in the D-STR, a finding that may be related to high-dose L-Dopa effects. In both regions, L-Dopa progressively attenuated reuptake kinetics through a decrease in Vmax and an increase in Km. This finding is consistent with recent clinical studies suggesting that L-Dopa chronically down-regulates the DA transporter (DAT), which may relate to the common development of L-Dopa induced dyskinesias (LID) in PD subjects. PMID:26611352

  3. Fast-scan cyclic voltammetry demonstrates that L-DOPA produces dose-dependent regionally selective, bimodal effects on striatal dopamine kinetics in vivo.

    PubMed

    Harun, R; Hare, K M; Brough, M E; Munoz, M J; Grassi, C M; Torres, G E; Grace, A A; Wagner, A K

    2015-11-27

    Parkinson's disease (PD) is a debilitating condition that is caused by a relatively specific degeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta. Levodopa (L-DOPA) was introduced as a viable treatment option for PD over 40 years ago and still remains the most common and effective therapy for PD. Though the effects of L-DOPA to augment striatal DA production are well known, little is actually known about how L-DOPA alters the kinetics of DA neurotransmission that contribute to its beneficial and adverse effects. In this study, we examined the effects of L-DOPA administration (50mg/kg carbidopa + 0, 100, and 250mg/kg L-DOPA) on regional electrically stimulated DA response kinetics using fast-scan cyclic voltammetry (FSCV) in anesthetized rats. We demonstrate that L-DOPA enhances DA release in both the dorsal striatum (D-STR) and nucleus accumbens (NAc), but surprisingly causes a delayed inhibition of release in the D-STR. In both regions, L-DOPA progressively attenuated reuptake kinetics, predominantly through a decrease in Vmax. These findings have important implications on understanding the pharmacodynamics of L-DOPA, which can be informative for understand its therapeutic effects and also common side effects like L-DOPA induced dyskinesias (LID). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. An Active Learning Mammalian Skeletal Muscle Lab Demonstrating Contractile and Kinetic Properties of Fast- and Slow-Twitch Muscle

    ERIC Educational Resources Information Center

    Head, S. I.; Arber, M. B.

    2013-01-01

    The fact that humans possess fast and slow-twitch muscle in the ratio of approximately 50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic…

  5. An Active Learning Mammalian Skeletal Muscle Lab Demonstrating Contractile and Kinetic Properties of Fast- and Slow-Twitch Muscle

    ERIC Educational Resources Information Center

    Head, S. I.; Arber, M. B.

    2013-01-01

    The fact that humans possess fast and slow-twitch muscle in the ratio of approximately 50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic…

  6. Effect of high loading on substrate utilization kinetics and microbial community structure in super fast submerged membrane bioreactor.

    PubMed

    Sözen, S; Çokgör, E U; Başaran, S Teksoy; Aysel, M; Akarsubaşı, A; Ergal, I; Kurt, H; Pala-Ozkok, I; Orhon, D

    2014-05-01

    The study investigated the effect of high substrate loading on substrate utilization kinetics, and changes inflicted on the composition of the microbial community in a superfast submerged membrane bioreactor. Submerged MBR was sequentially fed with a substrate mixture and acetate; its performance was monitored at steady-state, at extremely low sludge age values of 2.0, 1.0 and 0.5d, all adjusted to a single hydraulic retention time of 8.0 h. Each MBR run was repeated when substrate feeding was increased from 200 mg COD/L to 1000 mg COD/L. Substrate utilization kinetics was altered to significantly lower levels when the MBR was adjusted to higher substrate loadings. Molecular analysis of the biomass revealed that variable process kinetics could be correlated with parallel changes in the composition of the microbial community, mainly by a replacement mechanism, where newer species, better adapted to the new growth conditions, substituted others that are washed out from the system. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. LIGKA: A linear gyrokinetic code for the description of background kinetic and fast particle effects on the MHD stability in tokamaks

    SciTech Connect

    Lauber, Ph. Guenter, S.; Koenies, A.; Pinches, S.D.

    2007-09-10

    In a plasma with a population of super-thermal particles generated by heating or fusion processes, kinetic effects can lead to the additional destabilisation of MHD modes or even to additional energetic particle modes. In order to describe these modes, a new linear gyrokinetic MHD code has been developed and tested, LIGKA (linear gyrokinetic shear Alfven physics) [Ph. Lauber, Linear gyrokinetic description of fast particle effects on the MHD stability in tokamaks, Ph.D. Thesis, TU Muenchen, 2003; Ph. Lauber, S. Guenter, S.D. Pinches, Phys. Plasmas 12 (2005) 122501], based on a gyrokinetic model [H. Qin, Gyrokinetic theory and computational methods for electromagnetic perturbations in tokamaks, Ph.D. Thesis, Princeton University, 1998]. A finite Larmor radius expansion together with the construction of some fluid moments and specification to the shear Alfven regime results in a self-consistent, electromagnetic, non-perturbative model, that allows not only for growing or damped eigenvalues but also for a change in mode-structure of the magnetic perturbation due to the energetic particles and background kinetic effects. Compared to previous implementations [H. Qin, mentioned above], this model is coded in a more general and comprehensive way. LIGKA uses a Fourier decomposition in the poloidal coordinate and a finite element discretisation in the radial direction. Both analytical and numerical equilibria can be treated. Integration over the unperturbed particle orbits is performed with the drift-kinetic HAGIS code [S.D. Pinches, Ph.D. Thesis, The University of Nottingham, 1996; S.D. Pinches et al., CPC 111 (1998) 131] which accurately describes the particles' trajectories. This allows finite-banana-width effects to be implemented in a rigorous way since the linear formulation of the model allows the exchange of the unperturbed orbit integration and the discretisation of the perturbed potentials in the radial direction. Successful benchmarks for toroidal Alfven

  8. Stopped-flow kinetics of pH-responsive polyamine latexes: how fast is the latex-to-microgel transition?

    PubMed

    Morse, A J; Armes, S P; Mills, P; Swart, R

    2013-12-10

    Four poly(ethylene glycol)-stabilized polyamine latexes, namely, poly(2-vinylpyridine) (P2VP), poly(2-(tert-butylamino)ethyl methacrylate) (PTBAEMA), poly(2-(diethylamino)ethyl methacrylate) (PDEA), and poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) were prepared via emulsion copolymerization using divinylbenzene (DVB) as a cross-linker at 0.80 mol % for all formulations. According to dynamic light scattering studies, the resulting latexes were near-monodisperse and had approximately constant hydrodynamic diameters of 205-220 nm at pH 10; a latex-to-microgel transition was observed at around the respective pKa of each polyamine on addition of acid. The kinetics of swelling of each latex was investigated by the pH-jump method using a commercial stopped-flow instrument. The most rapid swelling was observed for the P2VP latex, which exhibited a characteristic swelling time (t*) of 5 ms. The corresponding t* values for PTBAEMA and PDEA were 25 and 35 ms, respectively, whereas the PDPA particles exhibited significantly slower swelling kinetics (t* = 180 ms). These t* values could not be correlated with either the latex Tg or the polyamine pKa. However, there is a positive correlation between t* and the repeat unit mass of the amine monomer, which suggests that the cationic charge density of the protonated polymer chains may influence the kinetics of swelling. Alternatively, the observed differences in swelling kinetics may simply reflect subtle differences in the DVB cross-link density, with more uniformly cross-linked latexes being capable of responding more quickly to a pH jump. The kinetics of deswelling for the corresponding microgel-to-latex transition was also briefly investigated for the PTBAEMA and P2VP particles. In both cases, much slower rates of deswelling were observed. This suggests that a latexlike "skin" is formed on the outer surface of the microgel particles during their deprotonation, which significantly retards the excretion of both salt and

  9. Angular distribution, kinetic energy distributions, and excitation functions of fast metastable oxygen fragments following electron impact of CO2

    NASA Technical Reports Server (NTRS)

    Misakian, M.; Mumma, M. J.; Faris, J. F.

    1975-01-01

    Dissociative excitation of CO2 by electron impact was studied using the methods of translational spectroscopy and angular distribution analysis. Earlier time of flight studies revealed two overlapping spectra, the slower of which was attributed to metastable CO(a3 pi) fragments. The fast peak is the focus of this study. Threshold energy, angular distribution, and improve time of flight measurements indicate that the fast peak actually consists of five overlapping features. The slowest of the five features is found to consist of metastable 0(5S) produced by predissociation of a sigma u + state of CO2 into 0(5S) + CO(a3 pi). Oxygen Rydberg fragments originating directly from a different sigma u + state are believed to make up the next fastest feature. Mechanisms for producing the three remaining features are discussed.

  10. A novel fast and flexible technique of radical kinetic behaviour investigation based on pallet for plasma evaluation structure and numerical analysis

    NASA Astrophysics Data System (ADS)

    Malinowski, Arkadiusz; Takeuchi, Takuya; Chen, Shang; Suzuki, Toshiya; Ishikawa, Kenji; Sekine, Makoto; Hori, Masaru; Lukasiak, Lidia; Jakubowski, Andrzej

    2013-07-01

    This paper describes a new, fast, and case-independent technique for sticking coefficient (SC) estimation based on pallet for plasma evaluation (PAPE) structure and numerical analysis. Our approach does not require complicated structure, apparatus, or time-consuming measurements but offers high reliability of data and high flexibility. Thermal analysis is also possible. This technique has been successfully applied to estimation of very low value of SC of hydrogen radicals on chemically amplified ArF 193 nm photoresist (the main goal of this study). Upper bound of our technique has been determined by investigation of SC of fluorine radical on polysilicon (in elevated temperature). Sources of estimation error and ways of its reduction have been also discussed. Results of this study give an insight into the process kinetics, and not only they are helpful in better process understanding but additionally they may serve as parameters in a phenomenological model development for predictive modelling of etching for ultimate CMOS topography simulation.

  11. In Situ TEM Observations of Sn-Containing Silicon Nanowires Undergoing Reversible Pore Formation Due to Fast Lithiation/Delithiation Kinetics

    SciTech Connect

    Lu, Xiaotang; Bogart, Timothy D.; Gu, Meng; Wang, Chong M.; Korgel, Brian

    2015-09-03

    In situ transmission electron microscopy (TEM) studies were carried out to observe directly in real time the lithiation and delithiation of silicon (Si) nanowires with significant amounts of tin (Sn). The incorporation of Sn significantly enhances the lithiation rate compared to typical Si nanowires. For instance, surface diffusion is enhanced by two orders of magnitude and the bulk lithiation rate by one order of magnitude, resulting in a sequential surface-then-core lithiation mechanism. Pore formation was observed in the nanowires during delithiation, most likely as a result of the fast delithiation kinetics of the nanowires. Pore formation was reversible and the pores disappeared during subsequent lithiation. When an amorphous Si shell was applied to the nanowires, pore formation was not observed during the in situ TEM experimences. Ex situ TEM analysis of Sn-containing Si nanowires cycled in coin cell batteries also showed that the application of an a-Si shell significantly retards pore formation in these nanowires.

  12. Fast Prediction of HCCI and PCCI Combustion with an Artificial Neural Network-Based Chemical Kinetic Model

    SciTech Connect

    Piggott, W T; Aceves, S M; Flowers, D L; Chen, J Y

    2007-09-26

    We have added the capability to look at in-cylinder fuel distributions using a previously developed ignition model within a fluid mechanics code (KIVA3V) that uses an artificial neural network (ANN) to predict ignition (The combined code: KIVA3V-ANN). KIVA3V-ANN was originally developed and validated for analysis of Homogeneous Charge Compression Ignition (HCCI) combustion, but it is also applicable to the more difficult problem of Premixed Charge Compression Ignition (PCCI) combustion. PCCI combustion refers to cases where combustion occurs as a nonmixing controlled, chemical kinetics dominated, autoignition process, where the fuel, air, and residual gas mixtures are not necessarily as homogeneous as in HCCI combustion. This paper analyzes the effects of introducing charge non-uniformity into a KIVA3V-ANN simulation. The results are compared to experimental results, as well as simulation results using a more physically representative and computationally intensive code (KIVA3V-MPI-MZ), which links a fluid mechanics code to a multi-zone detailed chemical kinetics solver. The results indicate that KIVA3V-ANN produces reasonable approximations to the more accurate KIVA3V-MPI-MZ at a much reduced computational cost.

  13. Ultrafine Nanocrystalline CeO2@C-Containing NaAlH4 with Fast Kinetics and Good Reversibility for Hydrogen Storage.

    PubMed

    Zhang, Xin; Liu, Yongfeng; Wang, Ke; Li, You; Gao, Mingxia; Pan, Hongge

    2015-12-21

    A nanocrystalline CeO2@C-containing NaAlH4 composite is successfully synthesized in situ by hydrogenating a NaH-Al mixture doped with CeO2@C. Compared with NaAlH4 , the as-prepared CeO2@C-containing NaAlH4 composite, with a minor amount of excess Al, exhibits significantly improved hydrogen storage properties. The dehydrogenation onset temperature of the hydrogenated [NaH-Al-7 wt % CeO2@C]-0.04Al sample is 77 °C lower than that of the pristine sample because of a reduced kinetic barrier. More importantly, the dehydrogenated sample absorbs ∼4.7 wt % hydrogen within 35 min at 100°C and 10 MPa of hydrogen. Compositional and structural analyses reveal that CeO2 is converted to CeH2 during ball milling and that the newly formed CeH2 works with the excess of Al to synergistically improve the hydrogen storage properties of NaAlH4. Our findings will aid in the rational design of novel catalyst-doped complex hydride systems with low operating temperatures, fast kinetics, and long-term cyclability.

  14. Similar mitochondrial activation kinetics in wild-type and creatine kinase-deficient fast-twitch muscle indicate significant Pi control of respiration.

    PubMed

    Jeneson, Jeroen A L; ter Veld, Frank; Schmitz, Joep P J; Meyer, Ronald A; Hilbers, Peter A J; Nicolay, Klaas

    2011-06-01

    Past simulations of oxidative ATP metabolism in skeletal muscle have predicted that elimination of the creatine kinase (CK) reaction should result in dramatically faster oxygen consumption dynamics during transitions in ATP turnover rate. This hypothesis was investigated. Oxygen consumption of fast-twitch (FT) muscle isolated from wild-type (WT) and transgenic mice deficient in the myoplasmic (M) and mitochondrial (Mi) CK isoforms (MiM CK(-/-)) were measured at 20°C at rest and during electrical stimulation. MiM CK(-/-) muscle oxygen consumption activation kinetics during a step change in contraction rate were 30% faster than WT (time constant 53 ± 3 vs. 69 ± 4 s, respectively; mean ± SE, n = 8 and 6, respectively). MiM CK(-/-) muscle oxygen consumption deactivation kinetics were 380% faster than WT (time constant 74 ± 4 s vs. 264 ± 4 s, respectively). Next, the experiments were simulated using a computational model of the oxidative ATP metabolic network in FT muscle featuring ADP and Pi feedback control of mitochondrial respiration (J. A. L. Jeneson, J. P. Schmitz, N. A. van den Broek, N. A. van Riel, P. A. Hilbers, K. Nicolay, J. J. Prompers. Am J Physiol Endocrinol Metab 297: E774-E784, 2009) that was reparameterized for 20°C. Elimination of Pi control via clamping of the mitochondrial Pi concentration at 10 mM reproduced past simulation results of dramatically faster kinetics in CK(-/-) muscle, while inclusion of Pi control qualitatively explained the experimental observations. On this basis, it was concluded that previous studies of the CK-deficient FT muscle phenotype underestimated the contribution of Pi to mitochondrial respiratory control.

  15. Measurement of kinetic energy release in CO fragmentation by charge-changing collisions of fast heavy ions

    SciTech Connect

    Mizuno, T.; Yamada, T.; Tsuchida, H.; Itoh, A.; Nakai, Y.

    2010-01-15

    We study ionization and fragmentation of CO in electron loss and capture collisions of B{sup 2+}, O{sup 2+}, and Si{sup 2+} ions at an energy of 71.4 keV/u (v=1.69 a.u.). Coincidence measurements of fragment ions from CO and charge-selected ions were performed by means of a momentum three-dimensional imaging technique. Production cross sections of CO{sup r+} and branching ratios into various fragmentation channels were obtained for r=1-4. We also measured kinetic energy release (KER) in individual fragmentation channels. The KER spectra for r<=2 are found to be different for electron loss and capture collisions, while the difference becomes small for r>=3. As a measure of the degree of molecular fragmentation, the magnitude of the binding energy of the relevant electronic states seems the important parameter both in loss and capture collisions.

  16. Kinetic Energy Release of the Singly and Doubly Charged Methylene Chloride Molecule: The Role of Fast Dissociation.

    PubMed

    Alcantara, K F; Rocha, A B; Gomes, A H A; Wolff, W; Sigaud, L; Santos, A C F

    2016-09-01

    The center of mass kinetic energy release distribution (KERD) spectra of selected ionic fragments, formed through dissociative single and double photoionization of CH2Cl2 at photon energies around the Cl 2p edge, were extracted from the shape and width of the experimentally obtained time-of-flight (TOF) distributions. The KERD spectra exhibit either smooth profiles or structures, depending on the moiety and photon energy. In general, the heavier the ionic fragments, the lower their average KERDs are. In contrast, the light H(+) fragments are observed with kinetic energies centered around 4.5-5.5 eV, depending on the photon energy. It was observed that the change in the photon energy involves a change in the KERDs, indicating different processes or transitions taking place in the breakup process. In the particular case of double ionization with the ejection of two charged fragments, the KERDs present have characteristics compatible with the Coulombic fragmentation model. Intending to interpret the experimental data, singlet and triplet states at Cl 2p edge of the CH2Cl2 molecule, corresponding to the Cl (2p → 10a1*) and Cl (2p → 4b1*) transitions, were calculated at multiconfigurational self-consistent field (MCSCF) level and multireference configuration interaction (MRCI). These states were selected to form the spin-orbit coupling matrix elements, which after diagonalization result in a spin-orbit manifold. Minimum energy pathways for dissociation of the molecule were additionally calculated aiming to give support to the presence of the ultrafast dissociation mechanism in the molecular breakup.

  17. Remobilizing surfactant retarded fluid particle interfaces. I. Stress-free conditions at the interfaces of micellar solutions of surfactants with fast sorption kinetics

    NASA Astrophysics Data System (ADS)

    Stebe, Kathleen J.; Lin, Shi-Yow; Maldarelli, Charles

    1991-01-01

    Surfactant molecules adsorb onto the interfaces of moving fluid particles and are convected to regions in which the surface flow converges. Accumulation of surfactant in these regions creates interfacial tension gradients that retard the surface flow. In this study it is argued theoretically and demonstrated experimentally that fluid movement on the surface of a drop or bubble can remain unhindered in the presence of a single adsorbed surfactant if, relative to the convective rate of transport of adsorbed surfactant along the surface, desorption is fast, and the bulk concentration is high enough so that diffusion away from the particle is fast. For this circumstance, a uniform surface concentration of surfactant is maintained, and no gradients in surface tension arise to retard the surface velocity. The fluid particle flow behaves as it would in the absence of surfactant save that it has a reduced, uniform surface tension. The remobilization of surfactant-laden interfaces of fluid particles is demonstrated experimentally in a three-phase periodic slug flow in a capillary tube in which a train of alternating air and aqueous slugs ride on an annular wetting film of fluorocarbon oil. Surfactant, dissolved in the aqueous slug phase, adsorbs onto and retards the aqueous-oil interface. The hydrodynamics of this flow is such that small changes in the mobility of this interface create large shear rates in the oil layer. This significantly increases the pressure drop required to drive the slug train at constant velocity. Three surface adsorbers are used to demonstrate surface remobilization: The polyethoxy, nonionic surfactants Triton X-100 and Brij-35, which have fast desorption kinetics and do not retard the surface flow at high concentrations and, as a counter example, the desorption hindered protein bovine serum albumin, which is shown to be unable to remobilize an interface even at high concentration.

  18. Mechanism for the formation of substituted manganese(V) cyanidonitrido complexes: crystallographic and kinetic study of the substitution reactions of trans-[MnN(H2O)(CN)4]2- with monodentate pyridine and bidentate pyridine-carboxylate ligands.

    PubMed

    van der Westhuizen, Hendrik J; Meijboom, Reinout; Schutte, Marietjie; Roodt, Andreas

    2010-10-18

    Dissolution of [(CH(3))N](2)Na[MnN(CN)(5)]·H(2)O in water results in the rapid dissociation of the trans-CN(-) ligand to form trans-[MnN(H(2)O)(CN)(4)](2-)(aq), which reacts with monodentate pyridine ligands such as 3-methyl and 4-methyl pyridine to form the corresponding mono-substituted complexes, of which the molecular structures obtained from X-ray crystallography, trans-[MnN(3-pic)(CN)(4)](2-) and trans-[MnN(4-pic)(CN)(4)](2-), are reported. [MnN(H(2)O)(CN)(4)](2-)(aq) also reacts with bidentate nucleophiles such as pyridine-2-carboxylate (pico) and quinoline-2-carboxylate (quino), yielding the corresponding [MnN(η(2)-pico)(CN)(3)](2-) and [MnN(η(2)-quino)(CN)(3)](2-) complexes as determined by X-ray crystallography. The formation kinetics of pyridine-2-carboxylate and three different pyridine-2,x-dicarboxylate ligands (x = 3, 4, 5) are reported, and two consecutive reaction steps are proposed, defined as the formation of the [MnN(η(1)-pico)(CN)(4)](3-) and [MnN(η(2)-pico)(CN)(3)](3-) complexes, respectively. Only the second steps could be spectrophotometrically observed and kinetically investigated. The first reaction is attributed to the rapid aqua substitution of [MnN(H(2)O)(CN)(4)](2-), thermodynamically unfavored and too fast to observe by conventional rapid third generation stopped-flow techniques. The second, slower reaction is attributed to cyanido substitution, with overall formation rate constants (25 °C; k(1)'; M(-1) s(-1)) and corresponding activation parameters (ΔH(k1')(double dagger), kJ mol(-1), ΔS(k1')(double dagger), J K(-1) mol(-1)) for the following entering bidentate nucleophiles: pyridine-2-carboxylate: (1.15 ± 0.04) × 10(-3), 102 ± 1, and 48 ± 3; pyridine-2,3-dicarboxylate: (1.1 ± 0.1) × 10(-3), 93 ± 2, and 20 ± 4; pyridine-2,4-dicarboxylate (8.5 ± 0.5) × 10(-4), 123 ± 5, and 115 ± 14; pyridine-2,5-dicarboxylate: (1.08 ± 0.04) × 10(-3), 106 ± 1, and 60 ± 2. A dissociative activation for the cyanido substitution

  19. Kv3.1/Kv3.2 channel positive modulators enable faster activating kinetics and increase firing frequency in fast-spiking GABAergic interneurons.

    PubMed

    Boddum, Kim; Hougaard, Charlotte; Xiao-Ying Lin, Julie; von Schoubye, Nadia Lybøl; Jensen, Henrik Sindal; Grunnet, Morten; Jespersen, Thomas

    2017-02-24

    Due to their fast kinetic properties, Kv3.1 voltage gated potassium channels are important in setting and controlling firing frequency in neurons and pivotal in generating high frequency firing of interneurons. Pharmacological activation of Kv3.1 channels may possess therapeutic potential for treatment of epilepsy, hearing disorders, schizophrenia and cognitive impairments. Here we thoroughly investigate the selectivity and positive modulation of the two small molecules, EX15 and RE01, on Kv3 channels. Selectivity studies, conducted in Xenopus laevis oocytes confirmed a positive modulatory effect of the two compounds on Kv3.1 and to a minor extent on Kv3.2 channels. RE01 had no effect on the Kv3.3 and Kv3.4 channels, whereas EX15 had an inhibitory impact on the Kv3.4 mediated current. Voltage-clamp experiments in monoclonal hKv3.1b/HEK293 cells (34 °C) revealed that the two compounds indeed induced larger currents and faster activation kinetics. They also decrease the speed of deactivation and shifted the voltage dependence of activation, to a more negative activation threshold. Application of action potential clamping and repetitive stimulation protocols of hKv3.1b expressing HEK293 cells revealed that EX15 and RE01 significantly increased peak amplitude, half width and decay time of Kv3.1 mediated currents, even during high-frequency action potential clamping (250 Hz). In rat hippocampal slices, EX15 and RE01 increased neuronal excitability in fast-spiking interneurons in dentate gyrus. Action potential frequency was prominently increased at minor depolarizing steps, whereas more marginal effects of EX15 and RE01 were observed after stronger depolarizations. In conclusion, our results suggest that EX15 and RE01 positive modulation of Kv3.1 and Kv3.2 currents facilitate increased firing frequency in fast-spiking GABAergic interneurons.

  20. Influence of the ligand alkyl chain length on the solubility, aqueous speciation, and kinetics of substitution reactions of water-soluble M3S4 (M = Mo, W) clusters bearing hydroxyalkyl diphosphines.

    PubMed

    Beltrán, Tomás F; Llusar, Rosa; Sokolov, Maxim; Basallote, Manuel G; Fernández-Trujillo, M Jesús; Pino-Chamorro, Jose Ángel

    2013-08-05

    Water-soluble [M3S4X3(dhbupe)3](+) diphosphino complexes (dhbupe = 1,2-bis(bis(hydroxybutyl)phosphino)ethane), 1(+) (M = Mo, X = Cl) and 2(+) (M = W; X = Br), have been synthesized by extending the procedure used for the preparation of their hydroxypropyl analogues by reaction of the M3S4(PPh3)3X4(solvent)x molecular clusters with the corresponding 1,2-bis(bishydroxyalkyl)diphosphine. The solid state structure of the [M3S4X3(dhbupe)3](+) cation possesses a C3 symmetry with a cuboidal M3S4 unit, and the outer positions are occupied by one halogen and two phosphorus atoms of the diphosphine ligand. At a basic pH, the halide ligands are substituted by hydroxo groups to afford the corresponding [Mo3S4(OH)3(dhbupe)3](+) (1OH(+)) and [W3S4(OH)3(dhbupe)3](+) (2OH(+)) complexes. This behavior is similar to that found in 1,2-bis(bis(hydroxymethyl)phosphino)ethane (dhmpe) complexes and differs from that observed for 1,2-bis(bis(hydroxypropyl)phosphino)ethane (dhprpe) derivatives. In the latter case, an alkylhydroxo group of the functionalized diphosphine replaces the chlorine ligands to afford Mo3S4 complexes in which the deprotonated dhprpe acts in a tridentate fashion. Detailed studies based on stopped-flow, (31)P{(1)H} NMR, and electrospray ionization mass spectrometry techniques have been carried out in order to understand the solution behavior and kinetics of interconversion between the different species formed in solution: 1 and 1OH(+) or 2 and 2OH(+). On the basis of the kinetic results, a mechanism with two parallel reaction pathways involving water and OH(-) attacks is proposed for the formal substitution of halides by hydroxo ligands. On the other hand, reaction of the hydroxo clusters with HX acids occurs with protonation of the OH(-) ligands followed by substitution of coordinated water by X(-).

  1. Water-mediated cation intercalation of open-framework indium hexacyanoferrate with high voltage and fast kinetics

    PubMed Central

    Chen, Liang; Shao, Hezhu; Zhou, Xufeng; Liu, Guoqiang; Jiang, Jun; Liu, Zhaoping

    2016-01-01

    Rechargeable aqueous metal-ion batteries made from non-flammable and low-cost materials offer promising opportunities in large-scale utility grid applications, yet low voltage and energy output, as well as limited cycle life remain critical drawbacks in their electrochemical operation. Here we develop a series of high-voltage aqueous metal-ion batteries based on ‘M+/N+-dual shuttles' to overcome these drawbacks. They utilize open-framework indium hexacyanoferrates as cathode materials, and TiP2O7 and NaTi2(PO4)3 as anode materials, respectively. All of them possess strong rate capability as ultra-capacitors. Through multiple characterization techniques combined with ab initio calculations, water-mediated cation intercalation of indium hexacyanoferrate is unveiled. Water is supposed to be co-inserted with Li+ or Na+, which evidently raises the intercalation voltage and reduces diffusion kinetics. As for K+, water is not involved in the intercalation because of the channel space limitation. PMID:27321702

  2. Water-mediated cation intercalation of open-framework indium hexacyanoferrate with high voltage and fast kinetics.

    PubMed

    Chen, Liang; Shao, Hezhu; Zhou, Xufeng; Liu, Guoqiang; Jiang, Jun; Liu, Zhaoping

    2016-06-20

    Rechargeable aqueous metal-ion batteries made from non-flammable and low-cost materials offer promising opportunities in large-scale utility grid applications, yet low voltage and energy output, as well as limited cycle life remain critical drawbacks in their electrochemical operation. Here we develop a series of high-voltage aqueous metal-ion batteries based on 'M(+)/N(+)-dual shuttles' to overcome these drawbacks. They utilize open-framework indium hexacyanoferrates as cathode materials, and TiP2O7 and NaTi2(PO4)3 as anode materials, respectively. All of them possess strong rate capability as ultra-capacitors. Through multiple characterization techniques combined with ab initio calculations, water-mediated cation intercalation of indium hexacyanoferrate is unveiled. Water is supposed to be co-inserted with Li(+) or Na(+), which evidently raises the intercalation voltage and reduces diffusion kinetics. As for K(+), water is not involved in the intercalation because of the channel space limitation.

  3. Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements.

    PubMed

    Gómez Ruiz, Braulio; Roux, Stéphanie; Courtois, Francis; Bonazzi, Catherine

    2016-11-15

    A simple, rapid and reliable method was developed for quantifying ascorbic (AA) and dehydroascorbic (DHAA) acids and validated in 20mM malate buffer (pH 3.8). It consists in a spectrophotometric measurement of AA, either directly on the solution added with metaphosphoric acid or after reduction of DHAA into AA by dithiothreitol. This method was developed with real time measurement of reactions kinetics in bulk reactors in mind, and was checked in terms of linearity, limits of detection and quantification, fidelity and accuracy. The linearity was found satisfactory on the range of 0-6.95mM with limits of detection and quantification of 0.236mM and 0.467mM, respectively. The method was found acceptable in terms of fidelity and accuracy with a coefficient of variation for repeatability and reproducibility below 6% for AA and below 15% for DHAA, and with a recovery range of 97-102% for AA and 88-112% for DHAA.

  4. Water-mediated cation intercalation of open-framework indium hexacyanoferrate with high voltage and fast kinetics

    NASA Astrophysics Data System (ADS)

    Chen, Liang; Shao, Hezhu; Zhou, Xufeng; Liu, Guoqiang; Jiang, Jun; Liu, Zhaoping

    2016-06-01

    Rechargeable aqueous metal-ion batteries made from non-flammable and low-cost materials offer promising opportunities in large-scale utility grid applications, yet low voltage and energy output, as well as limited cycle life remain critical drawbacks in their electrochemical operation. Here we develop a series of high-voltage aqueous metal-ion batteries based on `M+/N+-dual shuttles' to overcome these drawbacks. They utilize open-framework indium hexacyanoferrates as cathode materials, and TiP2O7 and NaTi2(PO4)3 as anode materials, respectively. All of them possess strong rate capability as ultra-capacitors. Through multiple characterization techniques combined with ab initio calculations, water-mediated cation intercalation of indium hexacyanoferrate is unveiled. Water is supposed to be co-inserted with Li+ or Na+, which evidently raises the intercalation voltage and reduces diffusion kinetics. As for K+, water is not involved in the intercalation because of the channel space limitation.

  5. Effects of hydraulic retention time on aerobic granulation and granule growth kinetics at steady state with a fast start-up strategy.

    PubMed

    Liu, Yong-Qiang; Zhang, Xing; Zhang, Rui; Liu, Wen-Tso; Tay, Joo-Hwa

    2016-01-01

    A hydraulic retention time (HRT) of 4, 6, and 8 h was employed, respectively, in three reactors to study the effects of HRT on granulation with a newly developed fast granulation strategy, i.e., a strategy by combining strong hydraulic selection pressure with high organic loading rate (OLR). Granules with clear boundary appeared within 24 h after reactor start-up and all reactors reached a pseudo steady state after 6-day operation. A 4-h HRT resulted in a relatively higher increasing rate in terms of granule size during granule development period, i.e., 208 μm day(-1), and the bigger granule size and the higher sludge volume index at the pseudo steady state. For HRT of 6 or 8 h, no obvious difference was observed. However, it was found that HRT influenced sludge retention time (SRT) and kinetics significantly. A HRT changing from 4 to 8 h led to an increased SRT from 3 to 21 days, a decreased observed specific biomass growth rate (μ obs) and an decreased observed biomass yield (Y obs) of stable granules from 0.37 to 0.062 days(-1), and 0.177 to 0.055 g MLVSS g(-1) COD, respectively. Both μ obs and Y obs had a linear relationship with the reciprocal of HRT. In addition, the great difference of microbial community between seed sludge, sludge retained in the reactors, and sludge washed out indicated a strong microbial selection for fast granulation within 24 h. However, during the granule development period from day 1 to 6, no more microbial selection was observed except an adjustment of microbial community. Little influence of HRT on microbial population in granular sludge indicated a minor role of HRT played for granulation with the fast start-up strategy adopted in this study. The results demonstrated that hydraulic selection pressure for granulation was mainly from short settling time, which led to strong microbial selection during the granulation period. Meanwhile, although HRT did not affect granulation with the fast start-up strategy, it played an

  6. Germanium as a Sodium Ion Battery Material: In Situ TEM Reveals Fast Sodiation Kinetics with High Capacity

    SciTech Connect

    Lu, Xiaotang; Adkins, Emily R.; He, Yang; Zhong, Li; Luo, Langli; Mao, Scott X.; Wang, Chong M.; Korgel, Brian A.

    2016-01-29

    Amorphous germanium (a-Ge) nanowires have great potential for application as anodes in Na-ion batteries. However, the Na-Ge reaction is much less studied and understood compared with other metal alloy anodes. Here, in situ transmission electron microscopy (TEM) is used to study the sodiation/desodiation behavior of a-Ge nanowires. Unexpectedly, our experiments revealed that a-Ge nanowires can be charged at a very fast rate and the final sodiation product, with over 300% volume expansion, is close to Na3Ge instead of NaGe which was considered as the ultimate sodiation state that Ge could achieve. Porous structure was observed in desodiation and, in contrast to delithiation, Na extraction is more likely to create pores in the nanowires due to the much larger radius of the Na ion. This porous structure has demonstrated excellent robustness upon cycling: it could recover flawlessly from the giant pores that were created during experimentation. These results show that the potential of a-Ge for Na-ion battery applications may have been previously underestimated.

  7. High quality reduced graphene oxide flakes by fast kinetically controlled and clean indirect UV-induced radical reduction

    NASA Astrophysics Data System (ADS)

    Flyunt, Roman; Knolle, Wolfgang; Kahnt, Axel; Halbig, Christian E.; Lotnyk, Andriy; Häupl, Tilmann; Prager, Andrea; Eigler, Siegfried; Abel, Bernd

    2016-03-01

    This work highlights a surprisingly simple and kinetically controlled highly efficient indirect method for the production of high quality reduced graphene oxide (rGO) flakes via UV irradiation of aqueous dispersions of graphene oxide (GO), in which the GO is not excited directly. While the direct photoexcitation of aqueous GO (when GO is the only light-absorbing component) takes several hours of reaction time at ambient temperature (4 h) leading only to a partial GO reduction, the addition of small amounts of isopropanol and acetone (2% and 1%) leads to a dramatically shortened reaction time by more than two orders of magnitude (2 min) and a very efficient and soft reduction of graphene oxide. This method avoids the formation of non-volatile species and in turn contamination of the produced rGO and it is based on the highly efficient generation of reducing carbon centered isopropanol radicals via the reaction of triplet acetone with isopropanol. While the direct photolysis of GO dispersions easily leads to degradation of the carbon lattice of GO and thus to a relatively low electric conductivity of the films of flakes, our indirect photoreduction of GO instead largely avoids the formation of defects, keeping the carbon lattice intact. Mechanisms of the direct and indirect photoreduction of GO have been elucidated and compared. Raman spectroscopy, XPS and conductivity measurements prove the efficiency of the indirect photoreduction in comparison with the state-of-the-art reduction method for GO (hydriodic acid/trifluoroacetic acid). The rapid reduction times and water solvent containing only small amounts of isopropanol and acetone may allow easy process up-scaling for technical applications and low-energy consumption.This work highlights a surprisingly simple and kinetically controlled highly efficient indirect method for the production of high quality reduced graphene oxide (rGO) flakes via UV irradiation of aqueous dispersions of graphene oxide (GO), in which the

  8. Role of the Fast Kinetics of Pyroglutamate-Modified Amyloid-β Oligomers in Membrane Binding and Membrane Permeability

    PubMed Central

    2015-01-01

    Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability. PMID:24950761

  9. High quality reduced graphene oxide flakes by fast kinetically controlled and clean indirect UV-induced radical reduction.

    PubMed

    Flyunt, Roman; Knolle, Wolfgang; Kahnt, Axel; Halbig, Christian E; Lotnyk, Andriy; Häupl, Tilmann; Prager, Andrea; Eigler, Siegfried; Abel, Bernd

    2016-04-14

    This work highlights a surprisingly simple and kinetically controlled highly efficient indirect method for the production of high quality reduced graphene oxide (rGO) flakes via UV irradiation of aqueous dispersions of graphene oxide (GO), in which the GO is not excited directly. While the direct photoexcitation of aqueous GO (when GO is the only light-absorbing component) takes several hours of reaction time at ambient temperature (4 h) leading only to a partial GO reduction, the addition of small amounts of isopropanol and acetone (2% and 1%) leads to a dramatically shortened reaction time by more than two orders of magnitude (2 min) and a very efficient and soft reduction of graphene oxide. This method avoids the formation of non-volatile species and in turn contamination of the produced rGO and it is based on the highly efficient generation of reducing carbon centered isopropanol radicals via the reaction of triplet acetone with isopropanol. While the direct photolysis of GO dispersions easily leads to degradation of the carbon lattice of GO and thus to a relatively low electric conductivity of the films of flakes, our indirect photoreduction of GO instead largely avoids the formation of defects, keeping the carbon lattice intact. Mechanisms of the direct and indirect photoreduction of GO have been elucidated and compared. Raman spectroscopy, XPS and conductivity measurements prove the efficiency of the indirect photoreduction in comparison with the state-of-the-art reduction method for GO (hydriodic acid/trifluoroacetic acid). The rapid reduction times and water solvent containing only small amounts of isopropanol and acetone may allow easy process up-scaling for technical applications and low-energy consumption.

  10. The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint.

    PubMed

    Papageorgiou, George C; Tsimilli-Michael, Merope; Stamatakis, Kostas

    2007-01-01

    The light-induced/dark-reversible changes in the chlorophyll (Chl) a fluorescence of photosynthetic cells and membranes in the mus-to-several min time window (fluorescence induction, FI; or Kautsky transient) reflect quantum yield changes (quenching/de-quenching) as well as changes in the number of Chls a in photosystem II (PS II; state transitions). Both relate to excitation trapping in PS II and the ensuing photosynthetic electron transport (PSET), and to secondary PSET effects, such as ion translocation across thylakoid membranes and filling or depletion of post-PS II and post-PS I pools of metabolites. In addition, high actinic light doses may depress Chl a fluorescence irreversibly (photoinhibitory lowering; q(I)). FI has been studied quite extensively in plants an algae (less so in cyanobacteria) as it affords a low resolution panoramic view of the photosynthesis process. Total FI comprises two transients, a fast initial (OPS; for Origin, Peak, Steady state) and a second slower transient (SMT; for Steady state, Maximum, Terminal state), whose details are characteristically different in eukaryotic (plants and algae) and prokaryotic (cyanobacteria) oxygenic photosynthetic organisms. In the former, maximal fluorescence output occurs at peak P, with peak M lying much lower or being absent, in which case the PSMT phases are replaced by a monotonous PT fluorescence decay. In contrast, in phycobilisome (PBS)-containing cyanobacteria maximal fluorescence occurs at M which lies much higher than peak P. It will be argued that this difference is caused by a fluorescence lowering trend (state 1 --> 2 transition) that dominates the FI pattern of plants and algae, and correspondingly by a fluorescence increasing trend (state 2 --> 1 transition) that dominates the FI of PBS-containing cyanobacteria. Characteristically, however, the FI pattern of the PBS-minus cyanobacterium Acaryochloris marina resembles the FI patterns of algae and plants and not of the PBS

  11. Ligand Migration and Binding in Myoglobin Mutant L29W

    NASA Astrophysics Data System (ADS)

    Nienhaus, G. Ulrich; Waschipky, Robert; Nienhaus, Karin; Minkow, Oleksandr; Ostermann, Andreas; Parak, Fritz G.

    2001-09-01

    Myoglobin, a small globular heme protein that binds gaseous ligands such as O2, CO, and NO reversibly at the heme iron, has for many years been a paradigm for studying the effects of structure and dynamics on protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. To observe structural changes induced by ligand dissociation, we have investigated carbonmonoxy myoglobin (MbCO) mutant L29W using time-resolved infrared spectroscopy in combination with x-ray crystallography. The presence of two distinct infrared stretch bands of the bound CO, AI at 1945 cm-1 and AII at 1955 cm-1, implies that L29W MbCO assumes two different conformations at neutral pH. Low-temperature flash photolysis experiments with monitoring of the absorption changes in the individual CO lines reveal markedly different rebinding properties. While recombination in AII is conceptually simple and well described by a two-state transition involving a distribution of enthalpy barriers, recombination in AI is more complicated: Besides a fast kinetic component, a second, slower kinetic component appears; its population grows with increasing temperature. X-ray crystallography of crystals illuminated below 180 K to photodissociate the CO reveals that the slow component arises from ligands that have migrated from their initial docking site to a remote site within the distal heme pocket. This process occurs in an essentially immobilized, frozen protein. Subsequently, ligands rebind by thermal activation over a barrier that is much higher than the barrier for recombination from the initial docking site. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. The x-ray structure shows a large proportion of ligands in a cavity on

  12. Crystal structures and DFT calculations of new chlorido-dimethylsulfoxide-M(III) (M = Ir, Ru, Rh) complexes with the N-pyrazolyl pyrimidine donor ligand: kinetic vs. thermodynamic isomers.

    PubMed

    Cánaves, María M; Cabra, María I; Bauzá, Antonio; Cañellas, Pablo; Sánchez, Kika; Orvay, Francisca; García-Raso, Angel; Fiol, Juan J; Terrón, Angel; Barceló-Oliver, Miquel; Ballester, Pablo; Mata, Ignasi; Molins, Elies; Hussain, Firasat; Frontera, Antonio

    2014-05-07

    New chlorido-dimethylsulfoxide-iridium(III), ruthenium(III) and rhodium(III) complexes with the 2-(1H-pyrazol-1-yl)-pyrimidine (pyrapyr) ligand (OC-6-N1)-[Rh(III)Cl3(DMSO-κS)(pyrapyr)] (1a, N = 3 and 1b, N = 4); (OC-6-N1)-[Ru(III)Cl3(DMSO-κS)(pyrapyr)] (2a, N = 3 and 2b, N = 4) and (OC-6-N1)-[Ir(III)Cl3(DMSO-κS)(pyrapyr)] (3a, N = 3 and 3b, N = 4) have been synthesized and characterized by spectroscopic techniques and by single crystal X-ray diffraction studies (1a, 1b, 2a, 2b, a disordered crystal 3a/3b and a cocrystal 3a·3b). In all cases, the metal centers show octahedral geometry coordinated to three chloride ligands and one S coordinated dimethylsulfoxide (DMSO-κS). The coordination sphere of the metal is completed by the pyrapyr molecule. Two different coordination modes are observed: (i) the DMSO-κS is opposite to the pyrimidinic N atom (IUPAC nomenclature is OC-6-31 denoted herein as trans); (ii) DMSO-κS is opposite to the pyrazolic N atom (IUPAC nomenclature is OC-6-41 denoted as cis). For Rh(III) the kinetic product (cis) yields the thermodynamic (trans) upon heating a solution of the kinetic product and both isomers have been X-ray characterized. Conversely for Ru(III), both kinetic and thermodynamic complexes have been obtained by using different procedures. Both isomers have been characterized by X-ray crystallography and the kinetic product does not yield the thermodynamic upon heating a solution of the former. Furthermore, the Ir(III) behaves differently, since both isomers are energetically equivalent and both isomers co-crystallize in the solid state. The kinetic/thermodynamic mechanism that yields the different isomers has been studied by using theoretical DFT calculations for each metal. Finally, two Ru(II) complexes (OC-6-N1)-[Ru(II)Cl2(DMSO-κS)2(pyrapyr)] (1a, N = 3 and 4b, N = 4) are also described and X-ray characterized. They were obtained as minor products during the synthesis of 2a.

  13. Hybrid Steered Molecular Dynamics Approach to Computing Absolute Binding Free Energy of Ligand-Protein Complexes: A Brute Force Approach That Is Fast and Accurate.

    PubMed

    Chen, Liao Y

    2015-04-14

    Computing the free energy of binding a ligand to a protein is a difficult task of essential importance for which purpose various theoretical/computational approaches have been pursued. In this paper, we develop a hybrid steered molecular dynamics (hSMD) method capable of resolving one ligand–protein complex within a few wall-clock days with high enough accuracy to compare with the experimental data. This hSMD approach is based on the relationship between the binding affinity and the potential of mean force (PMF) in the established literature. It involves simultaneously steering n (n = 1, 2, 3, ...) centers of mass of n selected segments of the ligand using n springs of infinite stiffness. Steering the ligand from a single initial state chosen from the bound state ensemble to the corresponding dissociated state, disallowing any fluctuations of the pulling centers along the way, one can determine a 3n-dimensional PMF curve connecting the two states by sampling a small number of forward and reverse pulling paths. This PMF constitutes a large but not the sole contribution to the binding free energy. Two other contributors are (1) the partial partition function containing the equilibrium fluctuations of the ligand at the binding site and the deviation of the initial state from the PMF minimum and (2) the partial partition function containing rotation and fluctuations of the ligand around one of the pulling centers that is fixed at a position far from the protein. We implement this hSMD approach for two ligand–protein complexes whose structures were determined and whose binding affinities were measured experimentally: caprylic acid binding to bovine β-lactoglobulin and glutathione binding to Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant. Our computed binding affinities agree with the experimental data within a factor of 1.5. The total time of computation for these two all-atom model systems (consisting of 96K and 114K atoms

  14. Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

    NASA Astrophysics Data System (ADS)

    Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R.

    2015-05-01

    Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using

  15. Fast quantitative ROS detection based on dual-color single rare-earth nanoparticle imaging reveals signaling pathway kinetics in living cells.

    PubMed

    Abdesselem, M; Ramodiharilafy, R; Devys, L; Gacoin, T; Alexandrou, A; Bouzigues, C I

    2017-01-05

    Reactive oxygen species (ROS), and notably hydrogen peroxide H2O2, are cellular second messengers that are known to control a variety of signaling processes. They can finely regulate the dynamics of signal transduction, cell response and ultimately tissue function. However, there are very few local, quantitative and time-resolved descriptions of their cellular organization at the scale of molecular reactions, due to the lack of efficient sensors. We thus developed a novel nanoprobe-based ROS detection system using the simultaneous imaging of single lanthanide nanoparticles (YAG:Ce and chemically reduced Gd0.6Eu0.4VO4). We reveal that both particle luminescence signals are controlled by their H2O2 local environment. By simultaneously tracking their luminescence, we devised a new approach providing a quantitative (0.5 μM accuracy in the 1-10 μM range) H2O2 measurement with a 500 ms time resolution, surpassing all existing methods by two orders of magnitude, and revealing previously inaccessible molecular events controlling ROS concentration. We used this nanoprobe in living cells to track fast signaling pathways, by measuring the dynamics of H2O2 intracellular concentrations, induced by endothelin-1 (ET-1) stimulation. We thus revealed the mechanisms controlling ROS production, notably the activity modulation of the ROS-producing enzyme NADPH oxidase by fast (<10 s) EGFR transactivation, and measured quantitatively their kinetic parameters through a minimal analytical model. Altogether, these results illustrate how lanthanide nanoparticle-based sensors are a powerful tool to dynamically probe molecular mechanisms shaping the oxidative cell response.

  16. Model of turnover kinetics in the lamellipodium: implications of slow- and fast- diffusing capping protein and Arp2/3 complex

    NASA Astrophysics Data System (ADS)

    McMillen, Laura M.; Vavylonis, Dimitrios

    2016-12-01

    Cell protrusion through polymerization of actin filaments at the leading edge of motile cells may be influenced by spatial gradients of diffuse actin and regulators. Here we study the distribution of two of the most important regulators, capping protein and Arp2/3 complex, which regulate actin polymerization in the lamellipodium through capping and nucleation of free barbed ends. We modeled their kinetics using data from prior single molecule microscopy experiments on XTC cells. These experiments have provided evidence for a broad distribution of diffusion coefficients of both capping protein and Arp2/3 complex. The slowly diffusing proteins appear as extended ‘clouds’ while proteins bound to the actin filament network appear as speckles that undergo retrograde flow. Speckle appearance and disappearance events correspond to assembly and dissociation from the actin filament network and speckle lifetimes correspond to the dissociation rate. The slowly diffusing capping protein could represent severed capped actin filament fragments or membrane-bound capping protein. Prior evidence suggests that slowly diffusing Apr2/3 complex associates with the membrane. We use the measured rates and estimates of diffusion coefficients of capping protein and Arp2/3 complex in a Monte Carlo simulation that includes particles in association with a filament network and diffuse in the cytoplasm. We consider two separate pools of diffuse proteins, representing fast and slowly diffusing species. We find a steady state with concentration gradients involving a balance of diffusive flow of fast and slow species with retrograde flow. We show that simulations of FRAP are consistent with prior experiments performed on different cell types. We provide estimates for the ratio of bound to diffuse complexes and calculate conditions where Arp2/3 complex recycling by diffusion may become limiting. We discuss the implications of slowly diffusing populations and suggest experiments to distinguish

  17. 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.

  18. Differences in zero-force and force-driven kinetics of ligand dissociation from beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy.

    PubMed

    Dettmann, W; Grandbois, M; André, S; Benoit, M; Wehle, A K; Kaltner, H; Gabius, H J; Gaub, H E

    2000-11-15

    Protein-carbohydrate interactions are involved in diverse regulatory processes. To help understand the mechanics and kinetics of dissociation of receptor-ligand complexes, we have analyzed the separation of lactose and the N-glycan chains of asialofetuin (ASF) from three lectins and an immunoglobulin G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)2 agglutinins from Ricinus communis (RCA) and Viscum album (VAA) show structural homology, the homodimeric galectin-1 from bovine heart (BHL) has no similarity to the two plant lectins except for sharing this monosaccharide specificity. The beta-galactoside-binding immunoglobulin G (IgG) fraction from human serum provides a further model system with distinct binding-site architecture. The k(off) constants for the two plant agglutinins were independent of the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0.5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing comparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/- 9 pN (RCA) were measured. For the same loading rate the rupture forces for the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupture force and the natural logarithm of the loading rate. Performing probability density and Monte Carlo calculations, the potential barrier widths, which determine the inverse dynamic dependence with the rate of force elevation, increased from 4 A (RCA) and 7 A (VAA and IgG) to 10 A (BHL) for the receptor-lactose interactions. Presenting ASF as ligand

  19. Decoding the Role of Water Dynamics in Ligand-Protein Unbinding: CRF1R as a Test Case.

    PubMed

    Bortolato, Andrea; Deflorian, Francesca; Weiss, Dahlia R; Mason, Jonathan S

    2015-09-28

    The residence time of a ligand-protein complex is a crucial aspect in determining biological effect in vivo. Despite its importance, the prediction of ligand koff still remains challenging for modern computational chemistry. We have developed aMetaD, a fast and generally applicable computational protocol to predict ligand-protein unbinding events using a molecular dynamics (MD) method based on adiabatic-bias MD and metadynamics. This physics-based, fully flexible, and pose-dependent ligand scoring function evaluates the maximum energy (RTscore) required to move the ligand from the bound-state energy basin to the next. Unbinding trajectories are automatically analyzed and translated into atomic solvation factor (SF) values representing the water dynamics during the unbinding event. This novel computational protocol was initially tested on two M3 muscarinic receptor and two adenosine A2A receptor antagonists and then evaluated on a test set of 12 CRF1R ligands. The resulting RTscores were used successfully to classify ligands with different residence times. Additionally, the SF analysis was used to detect key differences in the degree of accessibility to water molecules during the predicted ligand unbinding events. The protocol provides actionable working hypotheses that are applicable in a drug discovery program for the rational optimization of ligand binding kinetics.

  20. Sigma-1 receptor alters the kinetics of Kv1.3 voltage gated potassium channels but not the sensitivity to receptor ligands.

    PubMed

    Kinoshita, Maho; Matsuoka, Yoshikazu; Suzuki, Takeshi; Mirrielees, Jennifer; Yang, Jay

    2012-05-03

    Sigma1 receptors (Sigma1R) are intracellular chaperone proteins that bind psychotropic drugs and also clinically used drugs such as ketamine and haloperidol. Co-expression of the Sigma1R has been reported to enhance the sensitivity of several voltage-gated ion channels to Sigma1R ligands. Kv1.3 is the predominant voltage-gated potassium channel expressed in T lymphocytes with a documented role in immune activation. To gain a better understanding of Sigma1R modulation of Kv ion channels, we investigated the effects of Sigma1R co-expression on Kv1.3 physiology and pharmacology in ion channels expressed in Xenopus oocytes. We also explored the protein domains of Kv1.3 necessary for protein:protein interaction between Kv1.3 and Sigma1R through co-immunoprecipitation studies. Slowly inactivating outward-going currents consistent with Kv1.3 expression were elicited on step depolarizations. The current characterized by E(rev), V(1/2), and slope factor remained unchanged when co-expressed with Sigma1R. Analysis of inactivation time constant revealed a faster Kv1.3 current decay when co-expressed with Sigma1R. However the sensitivity to Sigma1R ligands remained unaltered when co-expressed with the Sigma1R in contrast to the previously reported modulation of ligand sensitivity in closely related Kv1.4 and Kv1.5 voltage gated potassium channels. Co-immunoprecipitation assays of various Kv1.3 truncation constructs indicated that the transmembrane domain of the Kv1.3 protein was responsible for the protein:protein interaction with the Sigma1R. Sigma1R likely interacts with different domains of Kv ion channel family proteins resulting in distinct modulation of different channels.

  1. Influence of equatorial and axial carboxylato ligands on the kinetic inertness of platinum(IV) complexes in the presence of ascorbate and cysteine and within DLD-1 cancer cells.

    PubMed

    Chen, Catherine K J; Zhang, Jenny Z; Aitken, Jade B; Hambley, Trevor W

    2013-11-14

    The rapid and premature reduction of platinum(IV) complexes in vivo is a significant impediment to these complexes being successfully employed as anticancer prodrugs. This study investigates the influence of the platinum(IV) coordination sphere on the ease of reduction of the platinum center in various biological contexts. In the presence of the biological reductants, ascorbate and cysteine, platinum(IV) complexes with dicarboxylato equatorial ligands were observed to exhibit lower reduction potentials and slower reduction rates than analogous platinum(IV) complexes with dichlorido equatorial ligands. Diaminetetracarboxylatoplatinum(IV) complexes exhibited unusually long half-lives in the presence of excess reductants; however, the complexes exhibited moderate potency in vitro, indicative of rapid reduction within the intracellular environment. By use of XANES spectroscopy, trans-[Pt(OAc)2(ox)(en)] and trans-[PtCl2(OAc)2(en)] were observed to be reduced at a similar rate within DLD-1 cancer cells. This large variability in kinetic inertness of diaminetetracarboxylatoplatinum(IV) complexes in different biological contexts has significant implications for the design of platinum(IV) prodrugs.

  2. Periodicity in tumor vasculature targeting kinetics of ligand-functionalized nanoparticles studied by dynamic contrast enhanced magnetic resonance imaging and intravital microscopy.

    PubMed

    Hak, Sjoerd; Cebulla, Jana; Huuse, Else Marie; Davies, Catharina de L; Mulder, Willem J M; Larsson, Henrik B W; Haraldseth, Olav

    2014-01-01

    In the past two decades advances in the development of targeted nanoparticles have facilitated their application as molecular imaging agents and targeted drug delivery vehicles. Nanoparticle-enhanced molecular imaging of the angiogenic tumor vasculature has been of particular interest. Not only because angiogenesis plays an important role in various pathologies, but also since endothelial cell surface receptors are directly accessible for relatively large circulating nanoparticles. Typically, nanoparticle targeting towards these receptors is studied by analyzing the contrast distribution on tumor images acquired before and at set time points after administration. Although several exciting proof-of-concept studies demonstrated qualitative assessment of relative target concentration and distribution, these studies did not provide quantitative information on the nanoparticle targeting kinetics. These kinetics will not only depend on nanoparticle characteristics, but also on receptor binding and recycling. In this study, we monitored the in vivo targeting kinetics of αvβ3-integrin specific nanoparticles with intravital microscopy and dynamic contrast enhanced magnetic resonance imaging, and using compartment modeling we were able to quantify nanoparticle targeting rates. As such, this approach can facilitate optimization of targeted nanoparticle design and it holds promise for providing more quantitative information on in vivo receptor levels. Interestingly, we also observed a periodicity in the accumulation kinetics of αvβ3-integrin targeted nanoparticles and hypothesize that this periodicity is caused by receptor binding, internalization and recycling dynamics. Taken together, this demonstrates that our experimental approach provides new insights in in vivo nanoparticle targeting, which may proof useful for vascular targeting in general.

  3. Monofunctional chorismate mutase from Bacillus subtilis: Kinetic and sup 13 C NMR studies on the interactions of the enzyme with its ligands

    SciTech Connect

    Gray, J.V.; Eren, D.; Knowles, J.R. )

    1990-09-18

    The interaction of the monofuctional chorismate mutase from Bacillus subtilis with chorismate and prephenate has been studied kinetically and by NMR spectroscopy with {sup 13}C specifically labeled substrates. Prephenate dominates the population of enzyme-bound species, and the off rate constant obtained from line-broadening experiments is close to the value of k{sub cat} for chorismate determined kinetically. The calculated on rate constant for prephenate is similar to the value of k{sub cat}/K{sub m} for chorismate. The kinetic parameters of the Bacillus mutase are remarkably insensitive to pH over a wide range and display no solvent isotope effect. These results suggest that the enzyme-catalyzed reaction may be encounter controlled (slowed from the diffusion limit by some feature of the enzyme's active site) and the k{sub cat} for chorismate is determined by the product off rate. There is now no evidence to suggest that the skeletal rearrangement on the enzyme surface occurs by a pathway other than a pericyclic process.

  4. Equilibrium and kinetic aspects of protein-DNA recognition.

    PubMed Central

    Livshitz, M A; Gursky, G V; Zasedatelev, A S; Volkenstein, M V

    1979-01-01

    The specificity of regulatory protein binding to DNA is due to a complementarity between the sequence of reaction centres on the protein and the base pair sequence in the specific DNA site allowing the formation of a number of specific noncovalent bonds between the interacting entities. In the present communication the thermodynamic and kinetic aspects of these interactions are considered. The extent of binding specificity is shown to increase with an increase of the bond stability constants and with an increase in the number of ligand reaction centres. Kinetic analysis is carried out assuming that association process is very fast and that dissociation of nonspecific complexes is a rate-limiting step in the recognition of a specific binding site on DNA. The calculations show that a ligand can recognize its specific binding site on DNA within a reasonably limited time interval if the number of its reaction centres and the corresponding stability constants are strongly limited. PMID:461187

  5. Equilibrium and kinetic aspects of protein-DNA recognition.

    PubMed

    Livshitz, M A; Gursky, G V; Zasedatelev, A S; Volkenstein, M V

    1979-01-01

    The specificity of regulatory protein binding to DNA is due to a complementarity between the sequence of reaction centres on the protein and the base pair sequence in the specific DNA site allowing the formation of a number of specific noncovalent bonds between the interacting entities. In the present communication the thermodynamic and kinetic aspects of these interactions are considered. The extent of binding specificity is shown to increase with an increase of the bond stability constants and with an increase in the number of ligand reaction centres. Kinetic analysis is carried out assuming that association process is very fast and that dissociation of nonspecific complexes is a rate-limiting step in the recognition of a specific binding site on DNA. The calculations show that a ligand can recognize its specific binding site on DNA within a reasonably limited time interval if the number of its reaction centres and the corresponding stability constants are strongly limited.

  6. Long Residence Time at the Neurosteroidogenic 18 kDa Translocator Protein Characterizes the Anxiolytic Ligand XBD173.

    PubMed

    Costa, Barbara; Da Pozzo, Eleonora; Cavallini, Chiara; Taliani, Sabrina; Da Settimo, Federico; Martini, Claudia

    2016-08-17

    Recent data have demonstrated a positive correlation between the residence time (RT) and neurosteroidogenic efficacy of a ligand at the translocator protein (TSPO), an attractive anxyolitic target. To explore the potential impact of RT on TSPO ligand anxiolytic activity, the RT and the steroidogenic activity of XBD173, a ligand exerting anxiolytic activity in humans, were retrospectively evaluated. To this aim, XBD173 association and dissociation rate constants were measured (1.23 × 10(7) M(-1) min(-1) and 0.0079 min(-1), respectively). XBD173 resulted to have a long RT (127 min) and to stimulate efficaciously neurosteroidogenesis, in terms of pregnenolone production. The present findings corroborate the importance of TSPO ligand RT to predict their effective neurosteroidogenic activity and promising anxiolytic action. These positive results prompted us to set up a fast and high-throughput kinetic method to improve the efficiency of RT-based TSPO drug-discovery process.

  7. Flow cytometry-based TCR-ligand Koff -rate assay for fast avidity screening of even very small antigen-specific T cell populations ex vivo.

    PubMed

    Nauerth, Magdalena; Stemberger, Christian; Mohr, Fabian; Weißbrich, Bianca; Schiemann, Matthias; Germeroth, Lothar; Busch, Dirk H

    2016-09-01

    High epitope-specific sensitivity of CD8(+) T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8(+) T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8(+) T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8(+) T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff -rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry.

  8. Single molecule high-throughput footprinting of small and large DNA ligands.

    PubMed

    Manosas, Maria; Camunas-Soler, Joan; Croquette, Vincent; Ritort, Felix

    2017-08-21

    Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.

  9. Kinetics of gravity-induced amyloplast sedimentation in statocytes of cress roots grown under fast clino-rotation, 1 g and after 180° inversion

    NASA Astrophysics Data System (ADS)

    Svegzdiene, D.; Rakleviciene, D.; Gaina, V.

    In order to detail the relation between the initial positioning of statoliths and root graviresponsiveness, the movement of amyloplasts within root statocytes of cress ( Lepidium sativum L.) seedlings grown under fast clino-rotation (50 rpm) and vertically at 1 g or 180° inverted after growth at 1 g has been analyzed during a subsequent 6-min period of lateral (reoriented 90°) gravistimulation. The main parameters of statolith motion kinetics such as relative positions and motion velocities in transverse and longitudinal directions have been evaluated taking linear measurements of organelle position with respect to the lower longitudinal and distal wall in the cells of the 2nd-5th columella storeys. A significant discrepancy in the direction and velocity of statolith movement has been determined within the first and second minutes of root horizontal placement. Centrally or proximally located statoliths in clino-rotated or inverted root statocytes moved transversely to the lower longitudinal cell wall more quickly as compared with distally located ones in 1- g root cells. At the same time, the longitudinal displacement of proximally located statoliths towards the cell center has been determined, while this displacement of distally located plastids was significantly more pronounced. The longitudinal motion of amyloplasts grouped in the central cell part was negligible within this 2-min period. After the 6th minute of stimulation, the total downward shift of the mean relative position of statoliths amounted to 16.3% from the total cell width in clino-rotated root statocytes and 17.1% in the cells of inverted roots, while it equalled 13.3% in 1- g root statocytes. The total change of the relative longitudinal position of amyloplasts with respect to the distal cell wall remained almost unchanged in roots grown on the clinostat. In inverted roots, the initial longitudinal position of 56.2% from the total cell length has been shifted significantly to 47.8% and from 27

  10. Flash kinetics in liquefied noble gases: Studies of alkane activation and ligand dynamics at rhodium carbonyl centers, and a search for xenon-carbene adducts

    SciTech Connect

    Yeston, Jake Simon

    2001-01-01

    A general introduction is given to place the subsequent chapters in context for the nonspecialist. Results are presented from a low temperature infrared (IR) flash kinetic study of C-H bond activation via photoinduced reaction of Cp*Rh(CO)2 (1) with linear and cyclic alkanes in liquid krypton and liquid xenon solution. No reaction was observed with methane; for all other hydrocarbons studied, the rate law supports fragmentation of the overall reaction into an alkane binding step followed by an oxidative addition step. For the binding step, larger alkanes within each series (linear and cyclic) interact more strongly than smaller alkanes with the Rh center. The second step, oxidative addition of the C-H bond across Rh, exhibits very little variance in the series of linear alkanes, while in the cyclic series the rate decreases with increasing alkane size. Results are presented from an IR flash kinetic study of the photoinduced chemistry of Tp*Rh(CO)2 (5; Tp* = hydridotris(3,5-dimethylpyrazolyl)borato) in liquid xenon solution at –50 °C. IR spectra of the solution taken 2 μs after 308 nm photolysis exhibit two transient bands at 1972-1980 cm-1 and 1992-2000 cm-1, respectively. These bands were assigned to (η3-Tp*)Rh(CO)•Xe and (η2-Tp*)Rh(CO)•Xe solvates on the basis of companion studies using Bp*Rh(CO)2 (9; Bp* = dihydridobis(3,5-dimethyl pyrazolyl)borato). Preliminary kinetic data for reaction of 5 with cyclohexane in xenon solution indicate that both transient bands still appear and that their rates of decay correlate with formation of the product Tp*Rh(CO)(C6H11)(H). The preparation and reactivity of the new complex Bp*Rh(CO)(pyridine) (11) are described. The complex reacts with CH3I to yield the novel Rh carbene hydride complex HB(Me2pz)2Rh(H)(I)(C5H5N)(C(O)Me) (12), resulting from formal addition of CH

  11. Mechanistic insights from resolving ligand-dependent kinetics of conformational changes at ATP-gated P2X1R ion channels

    PubMed Central

    Fryatt, Alistair G.; Dayl, Sudad; Cullis, Paul M.; Schmid, Ralf; Evans, Richard J.

    2016-01-01

    Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2′-deoxy ATP, 3′-deoxy ATP, Ap5A and αβmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2′ hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αβmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs. PMID:27616669

  12. Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: Pharmacological characterization of allosteric interactions with the sup 3 Hnitrobenzylthioinosine binding site

    SciTech Connect

    Hammond, J.R. )

    1991-06-01

    Kinetic analysis of the binding of {sup 3}Hnitrobenzylthioinosine ({sup 3}H NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of {sup 3}H NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the {sup 3}HNBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of {sup 3}HNBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of {sup 3}HNBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of {sup 3}HNBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of {sup 3}HNBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both {sup 3}HNBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of {sup 3}HNBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of {sup 3}HNBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of {sup 3}HNBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of {sup 3}HNBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam.

  13. Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targeting.

    PubMed

    Clausen, Thomas Mandel; Pereira, Marina Ayres; Oo, Htoo Zarni; Resende, Mafalda; Gustavson, Tobias; Mao, Yang; Sugiura, Nobuo; Liew, Janet; Fazli, Ladan; Theander, Thor G; Daugaard, Mads; Salanti, Ali

    2016-07-01

    In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.

  14. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    PubMed

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  15. A Novel Selective Inverse Agonist of the CB2 Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies.

    PubMed

    Martella, Andrea; Sijben, Huub; Rufer, Arne C; Grether, Uwe; Fingerle, Juergen; Ullmer, Christoph; Hartung, Thomas; IJzerman, Adriaan P; van der Stelt, Mario; Heitman, Laura H

    2017-10-01

    The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [(3)H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [(3)H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [(3)H]RO6957022 showed high affinity for human CB2R with fast association (kon) and moderate dissociation (koff) kinetics. To demonstrate the robustness of [(3)H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [(3)H]RO6957022 to study the kinetic binding profiles (i.e., kon and koff values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [(3)H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Geometric isomerism in pentacoordinate Cu2+ complexes: equilibrium, kinetic, and density functional theory studies reveal the existence of equilibrium between square pyramidal and trigonal bipyramidal forms for a tren-derived ligand.

    PubMed

    Algarra, Andrés G; Basallote, Manuel G; Castillo, Carmen E; Clares, M Paz; Ferrer, Armando; García-España, Enrique; Llinares, José M; Máñez, M Angeles; Soriano, Conxa

    2009-02-02

    A ligand (L1) (bis(aminoethyl)[2-(4-quinolylmethyl)aminoethyl]amine) containing a 4-quinolylmethyl group attached to one of the terminal amino groups of tris(2-aminoethyl)amine (tren) has been prepared, and its protonation constants and stability constants for the formation of Cu(2+) complexes have been determined. Kinetic studies on the formation of Cu(2+) complexes in slightly acidic solutions and on the acid-promoted complex decomposition strongly suggest that the Cu(2+)-L1 complex exists in solution as a mixture of two species, one of them showing a trigonal bipyramidal (tbp) coordination environment with an absorption maximum at 890 nm in the electronic spectrum, and the other one being square pyramidal (sp) with a maximum at 660 nm. In acidic solution only a species with tbp geometry is formed, whereas in neutral and basic solutions a mixture of species with tbp and sp geometries is formed. The results of density functional theory (DFT) calculations indicate that these results can be rationalized by invoking the existence of an equilibrium of hydrolysis of the Cu-N bond with the amino group supporting the quinoline ring so that CuL1(2+) would be actually a mixture of tbp [CuL1(H(2)O)](2+) and sp [CuL1(H(2)O)(2)](2+). As there are many Cu(2+)-polyamine complexes with electronic spectra that show two overlapping bands at wavelengths close to those observed for the Cu(2+)-L1 complex, the existence of this kind of equilibrium between species with two different geometries can be quite common in the chemistry of these compounds. A correlation found between the position of the absorption maximum and the tau parameter measuring the distortion from the idealized tbp and sp geometries can be used to estimate the actual geometry in solution of this kind of complex.

  17. Co(II), Ni(II), Cu(II) and Zn(II) complexes of tridentate ONO donor Schiff base ligand: Synthesis, characterization, thermal, non-isothermal kinetics and DFT calculations

    NASA Astrophysics Data System (ADS)

    Kusmariya, Brajendra S.; Mishra, A. P.

    2017-02-01

    We report here four mononuclear Co(II), Ni(II), Cu(II) and Zn(II) coordination compounds of general formula [M(L)2] {L = dcp; M = CoII, CuII & ZnII} and [M(L)(H2O)]·H2O {L = dcp; M = NiII} derived from tridentate 2,4-dichloro-6-{[(3-chloro-2-hydroxy-5-nitrophenyl)imino]methyl}phenol (dcp) ligand. These compounds were synthesized and characterized by elemental analysis, FT-IR, uv-vis, 1H NMR, molar conductance, magnetic moment, thermal, PXRD and SEM-EDX. The Powder X-ray Diffraction patterns and SEM analyses showed the crystalline nature of synthesized compounds. The peak broadening was explained in terms of crystallite size and the lattice strain using Scherrer and Williamson-Hall method. Thermogravimetric analysis was performed to determine the thermal stability of synthesized compounds under nitrogen atmosphere up to 820 K at 10 Kmin-1 heating rate. The kinetic and thermodynamic parameters of thermal decomposition were calculated using Coats-Redfern (C-R), Piloyan-Novikova (P-N) and Horowitz-Metzger (H-M) methods assuming first order degradation. The calculated optical band gap values of complexes were found to be in semiconducting range. To support the experimental findings, and derive some fruitful information viz. frequency calculations, HOMO-LUMO, energy gap (ΔE), molecular electrostatic potential (MEP), spin density, absorption spectra etc.; theoretical calculations by means of DFT and TD-DFT at B3LYP level were incorporated.

  18. Kinetic and Thermodynamic Stabilization of Metal Complexes by Introverted Coordination in a Calix[6]azacryptand.

    PubMed

    Inthasot, Alex; Brunetti, Emilio; Lejeune, Manuel; Menard, Nicolas; Prangé, Thierry; Fusaro, Luca; Bruylants, Gilles; Reinaud, Olivia; Luhmer, Michel; Jabin, Ivan; Colasson, Benoit

    2016-03-24

    The Huisgen thermal reaction between an organic azide and an acetylene was employed for the selective monofunctionalization of a X6 -azacryptand ligand bearing a tren coordinating unit [X6 stands for calix[6]arene and tren for tris(2-aminoethyl)amine]. Supramolecular assistance, originating from the formation of a host-guest inclusion complex between the reactants, greatly accelerates the reaction while self-inhibition affords a remarkable selectivity. The new ligand possesses a single amino-leg appended at the large rim of the calixarene core and the corresponding Zn(2+) complex was characterized both in solution and in the solid state. The coordination of Zn(2+) not only involves the tren cap but also the introverted amino-leg, which locks the metal ion in the cavity. Compared with the parent ligand deprived of the amino-leg, the affinity of the new monofunctionalized X6 tren ligand 6 for Zn(2+) is found to have a 10-fold increase in DMSO, which is a very competitive solvent, and with an enhancement of at least three orders of magnitude in CDCl3 /CD3 OD (1:1, v/v). In strong contrast with the fast binding kinetics, decoordination of Zn(2+) as well as transmetallation appeared to be very slow processes. The monofunctionalized X6 tren ligand 6 fully protects the metal ion from the external medium thanks to the combination of a cavity and a closed coordination sphere, leading to greater thermodynamic and kinetic stabilities.

  19. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay*

    PubMed Central

    AbuSamra, Dina B.; Al-Kilani, Alia; Hamdan, Samir M.; Sakashita, Kosuke; Gadhoum, Samah Z.; Merzaban, Jasmeen S.

    2015-01-01

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. PMID:26124272

  20. Exploring the Hydrolytic Behavior of the Platinum(IV) Complexes with Axial Acetato Ligands.

    PubMed

    Zhao, Jian; Xu, Zichen; Lin, Jing; Gou, Shaohua

    2017-08-21

    Platinum(IV) complexes are generally thought to be kinetically inert, and are expected to be stable enough to resist premature aquation before entering the cancer cells. Nevertheless, in this work, complex 2 with axial acetato ligands can hydrolyze relatively quickly under biologically relevant conditions with a half-life of 91.7 min, resulting in the loss of the equatorial chlorido ligand. Further study indicated that the fast hydrolysis of complex 2 may be attributed to the strong σ-donor ability of N-isopropyl-1R,2R-diaminocyclohexane, and an increasing σ-donor ability of the amine group can promote the hydrolysis rate of the corresponding platinum(IV) complex. The experiment results were proven by the corresponding DFT calculation. Our study can help to re-evaluate the aqueous properties of the platinum(IV) complexes with axial acetate, which may be less inert to hydrolysis than expected under biologically relevant conditions.

  1. Stability, water exchange, and anion binding studies on lanthanide(III) complexes with a macrocyclic ligand based on 1,7-diaza-12-crown-4: extremely fast water exchange on the Gd3+ complex.

    PubMed

    Pálinkás, Zoltán; Roca-Sabio, Adrián; Mato-Iglesias, Marta; Esteban-Gómez, David; Platas-Iglesias, Carlos; de Blas, Andrés; Rodríguez-Blas, Teresa; Tóth, Eva

    2009-09-21

    The picolinate-derivative ligand based on the 1,7-diaza-12-crown-4 platform (bp12c4(2-)) forms stable Ln(3+) complexes with stability constants increasing from the early to the middle lanthanides, then being relatively constant for the rest of the series (logK(LnL) = 16.81(0.06), 18.82(0.01), and 18.08(0.05) for Ln = La, Gd, and Yb, respectively). The complex formation is fast, allowing for direct potentiometric titrations to assess the stability constants. In the presence of Zn(2+), the dissociation of [Gd(bp12c4)](+) proceeds both via proton- and metal-assisted pathways, and in this respect, this system is intermediate between DTPA-type and macrocyclic, DOTA-type chelates, for which the dissociation is predominated by metal- or proton-assisted pathways, respectively. The Cu(2+) exchange shows an unexpected pH dependency, with the observed rate constants decreasing with increasing proton concentration. The rate of water exchange, assessed by (17)O NMR, is extremely high on the [Gd(bp12c4)(H(2)O)(q)](+) complex (k(ex)(298) = (2.20 +/- 0.15) x 10(8) s(-1)), and is in the same order of magnitude as for the Gd(3+) aqua ion (k(ex)(298) = 8.0 x 10(8) s(-1)). In aqueous solution, the [Gd(bp12c4)(H(2)O)(q)](+) complex is present in hydration equilibrium between nine-coordinate, monohydrated, and ten-coordinate, bishydrated species. We attribute the fast exchange to the hydration equilibrium and to the flexible nature of the inner coordination sphere. The large negative value of the activation entropy (DeltaS = -35 +/- 8 J mol(-1) K(-1)) points to an associative character for the water exchange and suggests that water exchange on the nine-coordinate, monohydrated species is predominant in the overall exchange. Relaxometric and luminescence measurements on the Gd(3+) and Eu(3+) analogues, respectively, indicate strong binding of endogenous anions such as citrate, hydrogencarbonate, or phosphate to [Ln(bp12c4)](+) complexes (K(aff) = 280 +/- 20 M(-1), 630 +/- 50 M(-1), and

  2. {sup 237}Np: Oxidation state in vivo and chelation by multidentate catecholat and hydroxypyridinonate ligands

    SciTech Connect

    Durbin, P.W.; Kullgren, B.; Allen, P.G.; Bucher, J.J.; Edelstein, N.M.; Shuh, D.K.; Xu, J.; Raymond, K.N. |

    1998-07-01

    Chemically, {sup 237}Np(V) is as toxic as U(VI), and radiologically, about as toxic as {sup 239}Pu. Depending on redox conditions in vivo, {sup 237}Np exists as weakly complexing Np(V) (NpO{sub 2}{sup +}) or as Np(IV), which forms complexes as stable as those of Pu(IV). Ten multidentate catecholate (CAM) and hydroxypyridinonate (HOPO) ligands with great affinity for Pu(IV) were compared with CaNa{sub 3}-DTPA for in vivo chelation of {sup 237}Np. Mice were injected intravenously with {sup 237}NpO{sub 2}Cl: those in a kinetic study were killed 1 to 2,880 min; in ligand studies, fed mice were injected intraperitoneally with a ligand 5, 60, or 1,440 min after {sup 237}Np(V), mice fasted for 16 h were gastrically intubated with a ligand 3 min after {sup 237}Np(V), and all were killed 24 h after ligand administration; tissues and excreta were radioanalyzed. Rapid plasma clearance and urinary excretion of {sup 237}Np(V) resemble U(VI); deposition and early retention in skeleton and liver resemble Pu(IV). The x-ray absorption near edge structure spectroscopy (XANES) spectra of femora of {sup 237}Np(V)-injected mice, compared with spectra of Np(V) and Np(IV) from reference solids, showed predominantly Np(IV). Significant in vivo {sup 237}Np chelation was obtained with all of the HOPO and CAM ligands injected at molar ratio 22; the HOPO ligands reduced {sup 237}Np in skeleton, liver, and other soft tissue, on average, to 72, 25, and 25% of control, respectively, while CaNa{sub 3}-DTPA was ineffective.

  3. Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics.

    PubMed

    Chen, Jiekai; Liu, Jing; Chen, You; Yang, Jiaqi; Chen, Jing; Liu, He; Zhao, Xiangjie; Mo, Kunlun; Song, Hong; Guo, Lin; Chu, Shilong; Wang, Deping; Ding, Ke; Pei, Duanqing

    2011-06-01

    The ectopic expression of several transcription factors can restore embryonic cell fate to cultured somatic cells and generate induced pluripotent stem cells (iPSCs), revealing a previously unknown pathway to pluripotency. However, this technology is currently limited by low efficiency, slow kinetics and multi-factorial requirement. Here we show that reprogramming can be improved and dramatically accelerated by optimizing culture conditions. First, we developed an optimized defined medium, iCD1, which allows Oct4/Sox2/Klf4 (OSK)-mediated reprogramming to achieve ultra-high efficiency (~10% at day 8). We also found that this optimized condition renders both Sox2 and Klf4 dispensable, although the elimination of these two factors leads to lower efficiency and slower kinetics. Our studies define a shortened route, both in timing and factor requirement, toward pluripotency. This new paradigm not only provides a rationale to further improve iPSC generation but also simplifies the conceptual understanding of reprogramming by defined factors.

  4. Rational optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells with ultra-high efficiency and fast kinetics

    PubMed Central

    Chen, Jiekai; Liu, Jing; Chen, You; Yang, Jiaqi; Chen, Jing; Liu, He; Zhao, Xiangjie; Mo, Kunlun; Song, Hong; Guo, Lin; Chu, Shilong; Wang, Deping; Ding, Ke; Pei, Duanqing

    2011-01-01

    The ectopic expression of several transcription factors can restore embryonic cell fate to cultured somatic cells and generate induced pluripotent stem cells (iPSCs), revealing a previously unknown pathway to pluripotency. However, this technology is currently limited by low efficiency, slow kinetics and multi-factorial requirement. Here we show that reprogramming can be improved and dramatically accelerated by optimizing culture conditions. First, we developed an optimized defined medium, iCD1, which allows Oct4/Sox2/Klf4 (OSK)-mediated reprogramming to achieve ultra-high efficiency (∼10% at day 8). We also found that this optimized condition renders both Sox2 and Klf4 dispensable, although the elimination of these two factors leads to lower efficiency and slower kinetics. Our studies define a shortened route, both in timing and factor requirement, toward pluripotency. This new paradigm not only provides a rationale to further improve iPSC generation but also simplifies the conceptual understanding of reprogramming by defined factors. PMID:21445094

  5. Competitive ligand exchange between Cu-humic acid complexes and methanobactin.

    PubMed

    Pesch, M-L; Hoffmann, M; Christl, I; Kraemer, S M; Kretzschmar, R

    2013-01-01

    Copper has been found to play a key role in the physiology of methanotrophic micro-organisms, and methane oxidation may critically depend on the availability of Cu. In natural environments, such as soils, sediments, peat bogs, and surface waters, the presence of natural organic matter (NOM) can control the bioavailability of Cu by forming strong metal complexes. To promote Cu acquisition, methanotrophs exude methanobactin, a ligand known to have a high affinity for Cu. In this study, the capability of methanobactin for Cu acquisition from NOM was investigated using humic acid (HA) as a model substance. The kinetics of ligand exchange between Cu-HA and methanobactin was observed by UV-vis spectroscopy, and the speciation of Cu bound to methanobactin was determined by size-exclusion chromatography coupled to an ICP-MS. The results showed that Cu was mobilized from HA by a fast ligand exchange reaction following a second-order rate law with first-order kinetics for both methanobactin and Cu-HA complexes. The reaction rates decreased with decreasing temperature. Equilibrium experiments indicated that methanobactin was not sorbed to HA and proved that methanobactin is competitive with HA for Cu binding by forming strong 1:1 Cu-methanobactin complexes. Consequently, our results demonstrate that methanobactin can efficiently acquire Cu in organic-rich environments. © 2012 Blackwell Publishing Ltd.

  6. Early postnatal kinetics of colostral immunoglobulin G absorption in fed and fasted piglets and developmental expression of the intestinal immunoglobulin G receptor.

    PubMed

    Cabrera, R; Lin, X; Ashwell, M; Moeser, A; Odle, J

    2013-01-01

    The transport of IgG across the epithelial barrier and into the circulation is achieved in part by the neonatal Fc receptor (FcRn), and this provides passive immunity to the neonate. The objective of this study was to determine the effect of time and feeding state on IgG absorption, intestinal morphology, and expression of IgG receptors in the first 24 h postbirth. Twenty newborn pigs were obtained immediately after birth and fitted with umbilical arterial catheters. Colostrum was manually collected from 12 lactating sows and centrifuged to produce defatted colostrum. Piglets were orally gavaged with 32 mL defatted colostrum per kilogram of BW (given in 2 doses 1 h apart) either at birth (0 h) or at 12 h postbirth under either fed (milk replacer) or fasted (saline solution) condition (n=5 per treatment). A fifth reference group (n=5) was euthanized at birth. Blood was collected every hour for the first 2 h immediately after the catheter was inserted and then every 4 h until 12 h (i.e., 0, 1, 2, 4, 8, and 12 h) for the treatment in which the defatted colostrum was given right after birth. For the treatment gavaged at 12 h postbirth, the sampling schedule was at 12, 13, 14, 16, 20, and 24 h. At 12 h postgavage, pigs were euthanized and jejunum tissues were collected for measurement of villi height, width, crypt depth, and gene expression of FcRn and β2-microglobulin (β2M) via reverse transcription PCR. Pig serum IgG concentration was determined by radial immunodiffusion. Data were analyzed according to a 2×2 factorial arrangement of treatments (0 h-fed, 0 h-fasted, 12 h-fed, and 12 h-fasted). There was no interaction between the time (age) of offering defatted colostrum (0 vs. 12 h) and nutritional state (fed vs. fasted) for any of the measurements, and there were no differences between fed and fasted pigs. Serum IgG concentrations increased progressively with time. Piglets offered defatted colostrum at 0 h had greater (P<0.05) overall IgG absorption and greater

  7. Thermodynamics and high-pressure kinetics of a fast carbon dioxide fixation reaction by a (2,6-pyridinedicarboxamidato-hydroxo)nickel(II) complex.

    PubMed

    Troeppner, O; Huang, D; Holm, R H; Ivanović-Burmazović, I

    2014-04-14

    The previously reported carbon dioxide fixation reaction by the planar terminal hydroxide complex [Ni(pyN2(Me2))(OH)](1-) in DMF has been further characterized by determination of the equilibrium constants K(eq)²⁹⁸ = 2.4 ± 0.2 × 10(5) M(-1) and K(eq)²²³ = 1.3 ± 0.1 × 10(7) M(-1), as well as the volume of activation for the CO2 binding (ΔV(on)(≠223) = -21 ± 3 cm(3) mol(-1)) and back decarboxylation (ΔV(off)(≠223) = -13 ± 1 cm(3) mol(-1)) by high-pressure kinetics. The data are consistent with an earlier DFT computation, including the probable nature of the transition state, and support designating the reaction as one of the most completely investigated carbon dioxide fixation reactions of any type.

  8. Kinetic method for the large-scale analysis of the binding mechanism of histone deacetylase inhibitors.

    PubMed

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-09-01

    Performing kinetic studies on protein ligand interactions provides important information on complex formation and dissociation. Beside kinetic parameters such as association rates and residence times, kinetic experiments also reveal insights into reaction mechanisms. Exploiting intrinsic tryptophan fluorescence a parallelized high-throughput Förster resonance energy transfer (FRET)-based reporter displacement assay with very low protein consumption was developed to enable the large-scale kinetic characterization of the binding of ligands to recombinant human histone deacetylases (HDACs) and a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes. For the binding of trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and two other SAHA derivatives to HDAH, two different modes of action, simple one-step binding and a two-step mechanism comprising initial binding and induced fit, were verified. In contrast to HDAH, all compounds bound to human HDAC1, HDAC6, and HDAC8 through a two-step mechanism. A quantitative view on the inhibitor-HDAC systems revealed two types of interaction, fast binding and slow dissociation. We provide arguments for the thesis that the relationship between quantitative kinetic and mechanistic information and chemical structures of compounds will serve as a valuable tool for drug optimization. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Kinetic modeling and fitting software for interconnected reaction schemes: VisKin.

    PubMed

    Zhang, Xuan; Andrews, Jared N; Pedersen, Steen E

    2007-02-15

    Reaction kinetics for complex, highly interconnected kinetic schemes are modeled using analytical solutions to a system of ordinary differential equations. The algorithm employs standard linear algebra methods that are implemented using MatLab functions in a Visual Basic interface. A graphical user interface for simple entry of reaction schemes facilitates comparison of a variety of reaction schemes. To ensure microscopic balance, graph theory algorithms are used to determine violations of thermodynamic cycle constraints. Analytical solutions based on linear differential equations result in fast comparisons of first order kinetic rates and amplitudes as a function of changing ligand concentrations. For analysis of higher order kinetics, we also implemented a solution using numerical integration. To determine rate constants from experimental data, fitting algorithms that adjust rate constants to fit the model to imported data were implemented using the Levenberg-Marquardt algorithm or using Broyden-Fletcher-Goldfarb-Shanno methods. We have included the ability to carry out global fitting of data sets obtained at varying ligand concentrations. These tools are combined in a single package, which we have dubbed VisKin, to guide and analyze kinetic experiments. The software is available online for use on PCs.

  10. Kinetic Modeling and Fitting Software for Inter-connected Reaction Schemes: VisKin

    PubMed Central

    Zhang, Xuan; Andrews, Jared N.; Pedersen, Steen E.

    2007-01-01

    Reaction kinetics for complex, highly-interconnected kinetic schemes are modeled using analytical solutions to a system of ordinary differential equations. The algorithm employs standard linear algebra methods that are implemented using MatLab functions in a Visual Basic interface. A graphical user interface for simple entry of reaction schemes facilitates comparison of a variety of reaction schemes. To ensure microscopic balance, graph theory algorithms are used to determine violations of thermodynamic cycle constraints. Analytical solutions based on linear differential equations result in fast comparisons of first order kinetic rates and amplitudes as a function of changing ligand concentrations. For analysis of higher order kinetics, we also implemented a solution using numerical integration. In order to determine rate constants from experimental data, fitting algorithms using the Levenberg-Marquardt algorithm or using Broyden-Fletcher-Goldfarb-Shanno (BFGS) methods were implemented that adjust rate constants to fit the model to imported data. We have included the ability to carry out global fitting of data sets obtained at varying ligand concentrations. These tools are combined in a single package, which we have dubbed VisKin, to guide and analyze kinetic experiments. The software is available online for use on PCs. PMID:17207764

  11. Characterizing ligand-microtubule binding by competition methods.

    PubMed

    Díaz, José Fernando; Buey, Rubén Martínez

    2007-01-01

    The knowledge of the thermodynamics and kinetics of drug-microtubule interaction is essential to understand the structure/affinity relationship of a given ligand family. When a ligand does not show an appropriate signal change (absorbance or fluorescence) upon binding, the extensive direct characterization of its binding affinities and kinetic rate constants of association and dissociation becomes a complex task. In those cases it is possible to obtain these parameters by competition of the ligand with a reference one of the same binding site that shows such change. Nevertheless, although the experimental setup of the competition measurements is easier, the treatment of the data is complex because simultaneous equilibrium/kinetic equations have to be solved. In this chapter, the taxoid-binding site of the microtubules will be used as an example to describe experimental competition and data analysis methods to determine the binding constants and kinetic rates of association and dissociation of ligands for microtubules.

  12. Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.

    PubMed

    Waas, William F; Rainey, Mark A; Szafranska, Anna E; Dalby, Kevin N

    2003-10-28

    Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.

  13. Fission Fragment Mass Distributions and Total Kinetic Energy Release of 235-Uranium and 238-Uranium in Neutron-Induced Fission at Intermediate and Fast Neutron Energies

    SciTech Connect

    Duke, Dana Lynn

    2015-11-12

    This Ph.D. dissertation describes a measurement of the change in mass distributions and average total kinetic energy (TKE) release with increasing incident neutron energy for fission of 235U and 238U. Although fission was discovered over seventy-five years ago, open questions remain about the physics of the fission process. The energy of the incident neutron, En, changes the division of energy release in the resulting fission fragments, however, the details of energy partitioning remain ambiguous because the nucleus is a many-body quantum system. Creating a full theoretical model is difficult and experimental data to validate existing models are lacking. Additional fission measurements will lead to higher-quality models of the fission process, therefore improving applications such as the development of next-generation nuclear reactors and defense. This work also paves the way for precision experiments such as the Time Projection Chamber (TPC) for fission cross section measurements and the Spectrometer for Ion Determination in Fission (SPIDER) for precision mass yields.

  14. A Xenon dielectric barrier discharge lamp (172nm) with a fast-pulse voltage driver: Influence of the voltage waveform on plasma kinetic issues and light output.

    NASA Astrophysics Data System (ADS)

    Carman, Robert; Ward, Barry; Mildren, Richard; Kane, Deborah

    2003-10-01

    An important class of vacuum-ultraviolet (VUV) excimer lamps based on high pressure rare-gas and rare-gas halogen mixtures utilize the dielectric barrier discharge (DBD) to generate a transient, non-equilibrium plasma that yields high electrical to VUV conversion efficiency. Recent interest has focussed on the use of pulsed voltage excitation techniques (rather than conventional AC sinusoidal waveforms) to alter the physical appearance of the DBD "micro-discharges" from filamentary (AC) to semi-diffuse, conical or homogeneous (pulsed), whilst at the same time dramatically improving the lamp performance and VUV efficiency^1,2. We report results from a combined experimental/computer modelling study of a short-pulse excited co-axial DBD Xe lamp to investigate the influence of the pulsed voltage waveform on the discharge structure, lamp performance, VUV output, and electrical efficiency. The underlying plasma kinetics issues relating to lamp performance, including parasitic collisional processes that act to quench key xenon species population densities, are examined in detail. ^1 Vollkommer F and Hitzschke, US patent 5604410 (1997) ^2 R.P.Mildren and R.J.Carman, J.Phys.D, 34, L1-L6 (2001)

  15. Probing the structure of the ligand binding cavity of lipocalins by fluorescence spectroscopy.

    PubMed

    Patel, R C; Lange, D; McConathy, W J; Patel, Y C; Patel, S C

    1997-06-01

    The lipocalin superfamily constitutes a phylogenetically conserved group of more than 40 proteins that function in the binding and transport of a variety of physiologically important ligands. Members of this family subserve diverse functions as carriers of retinoids (retinol binding protein), odorants (odorant binding proteins), chromophores (insecticyanin, INS), pheromones (aphrodisin) and sterols (apolipoprotein D, apoD). Despite the pivotal importance of the ligand binding function of these proteins, a suitable approach for characterizing the molecular determinants of such binding has not been available. In studies using three homogeneously purified lipocalins INS, beta-lactoglobulin (BLG) and human apoD, we find that the fluorescence reporter BIS (1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid) is an ideal candidate for use in rapid kinetic experiments and in fluorescence resonance energy transfer (FRET). These methods require only small amounts of reagents and yield molecular coordinates of the ligand binding cavity of lipocalins in solution that are in remarkably close agreement to those obtained from crystallographic work with solids. Extremely fast ligand binding dynamics is indicated.

  16. Kinetic Study of OH Reactions With n-Octane and n-Decane Using Relative Rate Combining with Discharge Fast Flow and Mass Spectrometer Technique

    SciTech Connect

    Li, Zhuangjie

    2004-03-31

    The combination of the relative rate method with the discharge fast flow/mass spectrometer technique (RR/DF/MS) has been developed to measure the rate constants for gas phase reactions involving OH radicals. The RR/DF/MS technique was used to measure the rate constant for reactions of the OH radical with n-octane (k3) and cyclohexane (k5) using 1,4-dioxane as a reference compound and with n-decane (k6) using n-octane and 1,4-dioxane as reference compounds. At 298 K, these rate constants were determined to be k3=(8.88 {+-} 0.31) x 10-12, k5=(6.95 {+-} 0.20) x 10-12 and k6=(1.38 {+-} 0.08) x 10-11 cm3 molecule-1 s-1, respectively, which are in very good agreement with those measured using different techniques. The features of the RR/DF/MS technique are discussed.

  17. High-temperature fast-flow-reactor kinetics study of the reaction AlO + CO/sub 2/. -->. AlO/sub 2/ + CO. Thermochemical implications

    SciTech Connect

    Rogowski, D.F.; English, A.J.; Fontijn, A.

    1986-04-10

    The title reaction has been studied in a high-temperature fast-flow reactor (HTFFR) at temperatures from 500 to 1300 K. Laser-induced fluorescence was used to monitor relative (AlO). k(T) was determined to be (2.5 +/- 1.3) x 10/sup -14/ exp((400 +/- 280)/T) cm/sup 3/ molecule/sup -1/ s/sup -1/ (confidence level > 95%). The reaction probably proceeds via an intermediate complex which preferentially dissociates to the reactants. The negative activation energy implies D(O-AlO) greater than or equal to D(O-CO) = 127 kcal mol/sup -1/, which is incompatible with the O-AlO dissociation energy obtained for AlO/sub 2/ from Al/sub 2/O/sub 3/ evaporation-mass spectrometry studies. It is argued that the latter AlO/sub 2/ may have a different structure from that of the present work. 19 references, 3 figures, 1 table.

  18. Tailored Ligand Acceleration of the Cu-Catalyzed Azide-Alkyne Cycloaddition Reaction: Practical and Mechanistic Implications

    PubMed Central

    Presolski, Stanislav I.; Hong, Vu; Cho, So-Hye; Finn, M.G.

    2010-01-01

    Tris(heterocyclemethyl)amines containing mixtures of 1,2,3-triazolyl, 2-benzimidazoyl, and 2-pyridyl components were prepared for ligand acceleration of the copper-catalyzed azide-alkyne cycloaddition reaction. Two classes of ligands were identified: those that give rise to high reaction rates in coordinating solvents but which inhibit the process when used in excess relative to copper, and those that provide for fast catalysis in water and are not inhibitory in excess. Several “mixed” ligands were identified that performed well under both types of conditions. Kinetics measurements as a function of ligand:metal ratio and catalyst concentration were found to be consistent with an active Cu2L formulation. Since strongly bound chelating agents are not always the most effective, achieving optimal rates requires an assessment of the potential donor molecules in the reaction mixture. Simple rules are provided to guide the user in the choice of effective ligands and reaction conditions to suit most classes of substrates, solvents, and concentrations. PMID:20863116

  19. Bis(methylpyridine)-EDTA derivative as a potential ligand for PET imaging: synthesis, complexation, and biological evaluation.

    PubMed

    Singh, Pooja; Aggarwal, Swati; Tiwari, Anjani K; Kumar, Vikas; Pratap, Ramendra; Chuttani, Krishna; Mishra, Anil K

    2014-12-01

    A novel transitional metal ligand derivatized from EDTA-conjugated 2-amino-4-methyl pyridine, an acyclic vehicle (EDTA-Mepy2 ) was designed, synthesized, and characterized for PET imaging with ⁶⁸Ga. The drug likeliness and appropriate lipophilicity were first analyzed by molecular docking studies which shows interactive property of ligand with serum albumin protein (HSA: PDB 1E78), at Lys199, Arg257, and His242 residues, which make it more appropriate in transportation as a specific ligand for PET imaging. As a confirmation, binding constant of the ligand with human serum albumin was calculated at λex = 350 nm which was found to be 4.9 × 10³ m⁻¹. The pharmacokinetics of (68) Ga-EDTA-Mepy2 was analyzed by blood kinetics (t(1/2) slow: 3 h 56 min and t(1/2) fast: 32 min) and biodistribution (maximum % ID/g was found in kidney at 1 h). Further the capability of this ligand was analyzed as optical marker also, by recording λex = 380 nm, RFU = 8000; 710 nm, RFU = 1000 units at fixed λem = 280 nm. Additionally, in physiological conditions where its stability was calculated, suggests 15-20 times selectivity over the endogenously present metal ions (KG aL /KZ nL = 14.3, KG aL /KC uL = 18.1).

  20. Ligand-modified metal clusters for gas separation and purification

    DOEpatents

    Okrut, Alexander; Ouyang, Xiaoying; Runnebaum, Ron; Gates, Bruce C.; Katz, Alexander

    2017-02-21

    Provided is an organic ligand-bound metal surface that selects one gaseous species over another. The species can be closely sized molecular species having less than 1 Angstrom difference in kinetic diameter. In one embodiment, the species comprise carbon monoxide and ethylene. Such organic ligand-bound metal surfaces can be successfully used in gas phase separations or purifications, sensing, and in catalysis.

  1. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

    PubMed Central

    Reddish, Michael J.; Vaughn, Morgan B.; Fu, Rong; Dyer, R. Brian

    2016-01-01

    Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not. PMID:26901612

  2. Protein-ligand-based pharmacophores: generation and utility assessment in computational ligand profiling.

    PubMed

    Meslamani, Jamel; Li, Jiabo; Sutter, Jon; Stevens, Adrian; Bertrand, Hugues-Olivier; Rognan, Didier

    2012-04-23

    Ligand profiling is an emerging computational method for predicting the most likely targets of a bioactive compound and therefore anticipating adverse reactions, side effects and drug repurposing. A few encouraging successes have already been reported using ligand 2-D similarity searches and protein-ligand docking. The current study describes the use of receptor-ligand-derived pharmacophore searches as a tool to link ligands to putative targets. A database of 68,056 pharmacophores was first derived from 8,166 high-resolution protein-ligand complexes. In order to limit the number of queries, a maximum of 10 pharmacophores was generated for each complex according to their predicted selectivity. Pharmacophore search was compared to ligand-centric (2-D and 3-D similarity searches) and docking methods in profiling a set of 157 diverse ligands against a panel of 2,556 unique targets of known X-ray structure. As expected, ligand-based methods outperformed, in most of the cases, structure-based approaches in ranking the true targets among the top 1% scoring entries. However, we could identify ligands for which only a single method was successful. Receptor-ligand-based pharmacophore search is notably a fast and reliable alternative to docking when few ligand information is available for some targets. Overall, the present study suggests that a workflow using the best profiling method according to the protein-ligand context is the best strategy to follow. We notably present concrete guidelines for selecting the optimal computational method according to simple ligand and binding site properties.

  3. The Positive Effects of Priming Exercise on Oxygen Uptake Kinetics and High-Intensity Exercise Performance Are Not Magnified by a Fast-Start Pacing Strategy in Trained Cyclists

    PubMed Central

    Caritá, Renato Aparecido Corrêa; Greco, Camila Coelho; Denadai, Benedito Sérgio

    2014-01-01

    The purpose of this study was to determine both the independent and additive effects of prior heavy-intensity exercise and pacing strategies on the VO2 kinetics and performance during high-intensity exercise. Fourteen endurance cyclists (VO2max  = 62.8±8.5 mL.kg−1.min−1) volunteered to participate in the present study with the following protocols: 1) incremental test to determine lactate threshold and VO2max; 2) four maximal constant-load tests to estimate critical power; 3) six bouts of exercise, using a fast-start (FS), even-start (ES) or slow-start (SS) pacing strategy, with and without a preceding heavy-intensity exercise session (i.e., 90% critical power). In all conditions, the subjects completed an all-out sprint during the final 60 s of the test as a measure of the performance. For the control condition, the mean response time was significantly shorter (p<0.001) for FS (27±4 s) than for ES (32±5 s) and SS (32±6 s). After the prior exercise, the mean response time was not significantly different among the paced conditions (FS = 24±5 s; ES = 25±5 s; SS = 26±5 s). The end-sprint performance (i.e., mean power output) was only improved (∼3.2%, p<0.01) by prior exercise. Thus, in trained endurance cyclists, an FS pacing strategy does not magnify the positive effects of priming exercise on the overall VO2 kinetics and short-term high-intensity performance. PMID:24740278

  4. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design.

    PubMed

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M

    2015-09-09

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.

  5. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

    PubMed

    Kopacz, Malgorzata M; Heuts, Dominic P H M; Fraaije, Marco W

    2014-10-01

    Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavin-containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre-steady-state kinetic analyses of the enzyme with the use of single- and double-mixing stopped-flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half-reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD. The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product-mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady-state rate equations derived for ping-pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme-product complex.

  6. Dynamics of Catalytic Resolution of 2-Lithio-N-Boc-piperidine by Ligand Exchange

    PubMed Central

    Beng, Timothy K.; Tyree, William S.; Parker, Trent; Su, Chicheung; Williard, Paul G.

    2012-01-01

    The dynamics of the racemization, catalytic and stoichiometric dynamic resolution of 2-lithio-N-Boc-piperidine, 7, have been investigated. The kinetic order in TMEDA, for both racemization and resolution of the title compound, and the kinetic order in resolving ligands, have been determined. The catalytic dynamic resolution is 0.5-order in chiral ligand 8, 0.265 order in chiral ligand 10, and second order in TMEDA. The X-ray crystal structure of ligand 10 shows it to be an octamer. Dynamic NMR studies of the resolution process were obtained. Some of the requirements for a successful catalytic dynamic resolution by ligand exchange have been identified. PMID:22967289

  7. Multiple geminate ligand recombinations in human hemoglobin.

    PubMed Central

    Esquerra, R M; Goldbeck, R A; Reaney, S H; Batchelder, A M; Wen, Y; Lewis, J W; Kliger, D S

    2000-01-01

    The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences. PMID:10827999

  8. Hydrophobic protein-ligand interactions preserved in the gas phase.

    PubMed

    Liu, Lan; Bagal, Dhanashri; Kitova, Elena N; Schnier, Paul D; Klassen, John S

    2009-11-11

    The results of time-resolved thermal dissociation measurements and molecular dynamic simulations are reported for gaseous deprotonated ions of the specific complexes of bovine beta-lactoglobulin (Lg) and a series of the fatty acids (FA): CH(3)(CH(2))(x)COOH, where x = 10, 12, 14, and 16. At the reaction temperatures investigated, 25-66 degrees C, the gaseous ions dissociate exclusively by the loss of neutral FA. According to the kinetic data, and confirmed by ion mobility measurements, the (Lg + FA)(7-) ions exist in two, noninterconverting structures designated the fast (Lg + FA)(f)(7-) and slow (Lg + FA)(s)(7-) components. The Arrhenius parameters for both components are sensitive to the length of the FA aliphatic chain. For the fast components, the activation energy (E(a)) increases in a nearly linear fashion, with each methylene group contributing approximately 0.8 kcal mol(-1) to E(a). This is similar to the contribution of -CH(2)- groups to the solvation of n-alkanes in nonpolar solvents. Furthermore, the magnitude of the E(a) values for the fast components is similar to the solvation enthalpies expected for the FA aliphatic chains in nonpolar and weakly polar solvents. The E(a) values determined for the slow components are larger than those of the fast components. Furthermore, the E(a) values do not vary in a simple fashion with the length of the aliphatic chain. Molecular dynamics simulations performed on the (Lg + PA) complex revealed that, depending on the charge configuration, the (Lg + PA)(7-) ion can exist in two distinct structures, which differ primarily by the position of the EF loop. In the open structure the EF loop is positioned away from the entrance to the hydrophobic cavity and the ligand is stabilized only through nonpolar intermolecular interactions. In the closed structure the EF loop covers the entrance of the cavity and the carboxylic group of PA participates in H-bonds with residues on the EF loop or residues located at the entrance of

  9. Reversible sequential-binding probe receptor-ligand interactions in single cells.

    PubMed

    Schreiter, Christoph; Gjoni, Marinela; Hovius, Ruud; Martinez, Karen L; Segura, Jean-Manuel; Vogel, Horst

    2005-12-01

    With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association-dissociation kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition-binding assays using fluorescent derivatives of the antagonist alpha-conotoxin GI (alpha-CnTx). Thereby, we determined the binding constants of unlabeled alpha-CnTx and d-tubocurarine. The high selectivity of alpha-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells.

  10. Efficient chemoenzymatic synthesis of chiral pincer ligands.

    PubMed

    Felluga, Fulvia; Baratta, Walter; Fanfoni, Lidia; Pitacco, Giuliana; Rigo, Pierluigi; Benedetti, Fabio

    2009-05-01

    Chiral, nonracemic pincer ligands based on the 6-phenyl-2-aminomethylpyridine and 2-aminomethylbenzo[h]quinoline scaffolds were obtained by a chemoenzymatic approach starting from 2-pyridyl and 2-benzoquinolyl ethanone. In the enantiodifferentiating step, secondary alcohols of opposite absolute configuration were obtained by a baker's yeast reduction of the ketones and by lipase-mediated dynamic kinetic resolution of the racemic alcohols. Their transformation into homochiral 1-methyl-1-heteroarylethanamines occurred without loss of optical purity, giving access to pincer ligands used in enantioselective catalysis.

  11. IL-6 cooperates with peroxisome proliferator-activated receptor-α-ligands to induce liver fatty acid binding protein (LFABP) up-regulation.

    PubMed

    Vida, Margarita; Serrano, Antonia; Romero-Cuevas, Miguel; Pavón, Francisco J; González-Rodriguez, Agueda; Gavito, Ana L; Cuesta, Antonio L; Valverde, Angela M; Rodríguez de Fonseca, Fernando; Baixeras, Elena

    2013-08-01

    LFABP plays a critical role in the uptake and intracellular transport of fatty acids (FA) and other peroxisome proliferator-activated receptor alpha (PPARα) ligands. PPARα activation by PPARα ligands bound to LFABP results in gene expression of FA oxidation enzymes and de novo LFABP. The cytokine IL-6 is involved in regulating liver lipid oxidation. To study the ability of IL-6 to modulate the expression of the LFABP in hepatocytes. HepG2 and mouse primary hepatocytes were used to test LFABP mRNA and protein expression after IL-6 and PPARα-ligand treatments. Mice lacking IL-6 and wild-type C57Bl/6 were subjected to a fasting/re-feeding cycle to monitor hepatic LFABP mRNA kinetics after food intake. In hepatocyte cultures, IL-6 treatment stimulated a LFABP mRNA sustained expression. Combined treatment of IL-6 plus PPARα ligands further enhanced LFABP gene and protein expression. In contrast, pretreatment with the PPARα-antagonist GW-6471 prevented the up-regulation of LFABP mRNA induced by IL-6 in the late phase of LFABP kinetics. Furthermore, the up-regulation of LFABP mRNA observed in the liver of wild-type mice 8 h after re-feeding was absent in mice lacking IL-6. IL-6 induces LFABP kinetics in hepatocytes and is partially dependent on PPARα. The maximum increase in LFABP expression occurs when the stimulation with IL-6 and PPARα-ligands takes place simultaneously. The in vivo results indicate a postprandial regulation of LFABP that correlates with the presence of IL-6. These effects may have important implications in the postprandial increase in FA uptake and intracellular trafficking in the liver. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Kinetic Atom.

    ERIC Educational Resources Information Center

    Wilson, David B.

    1981-01-01

    Surveys the research of scientists like Joule, Kelvin, Maxwell, Clausius, and Boltzmann as it comments on the basic conceptual issues involved in the development of a more precise kinetic theory and the idea of a kinetic atom. (Author/SK)

  13. Kinetic Atom.

    ERIC Educational Resources Information Center

    Wilson, David B.

    1981-01-01

    Surveys the research of scientists like Joule, Kelvin, Maxwell, Clausius, and Boltzmann as it comments on the basic conceptual issues involved in the development of a more precise kinetic theory and the idea of a kinetic atom. (Author/SK)

  14. Stretchable Self-Healing Polymeric Dielectrics Cross-Linked Through Metal-Ligand Coordination.

    PubMed

    Rao, Ying-Li; Chortos, Alex; Pfattner, Raphael; Lissel, Franziska; Chiu, Yu-Cheng; Feig, Vivian; Xu, Jie; Kurosawa, Tadanori; Gu, Xiaodan; Wang, Chao; He, Mingqian; Chung, Jong Won; Bao, Zhenan

    2016-05-11

    A self-healing dielectric elastomer is achieved by the incorporation of metal-ligand coordination as cross-linking sites in nonpolar polydimethylsiloxane (PDMS) polymers. The ligand is 2,2'-bipyridine-5,5'-dicarboxylic amide, while the metal salts investigated here are Fe(2+) and Zn(2+) with various counteranions. The kinetically labile coordination between Zn(2+) and bipyridine endows the polymer fast self-healing ability at ambient condition. When integrated into organic field-effect transistors (OFETs) as gate dielectrics, transistors with FeCl2 and ZnCl2 salts cross-linked PDMS exhibited increased dielectric constants compared to PDMS and demonstrated hysteresis-free transfer characteristics, owing to the low ion conductivity in PDMS and the strong columbic interaction between metal cations and the small Cl(-) anions which can prevent mobile anions drifting under gate bias. Fully stretchable transistors with FeCl2-PDMS dielectrics were fabricated and exhibited ideal transfer characteristics. The gate leakage current remained low even after 1000 cycles at 100% strain. The mechanical robustness and stable electrical performance proved its suitability for applications in stretchable electronics. On the other hand, transistors with gate dielectrics containing large-sized anions (BF4(-), ClO4(-), CF3SO3(-)) displayed prominent hysteresis due to mobile anions drifting under gate bias voltage. This work provides insights on future design of self-healing stretchable dielectric materials based on metal-ligand cross-linked polymers.

  15. The Dynamics of Ligand Barrier Crossing Inside the Acetylcholinesterase Gorge

    SciTech Connect

    Bui, Jennifer M.; Henchman, Richard H.; Mccammon, Andy

    2003-10-01

    The dynamics of ligand movement through the constricted region of the acetylcholinesterase gorge is important in understanding how the ligand gains access to and is released from the active site of the enzyme. Molecular dynamics simulations of the simple ligand, tetramethylammonium, crossing this bottleneck region are conducted using umbrella potential sampling and activated .ux techniques. The low potential of mean force obtained is consistent with the fast reaction rate of acetylcholinesterase observed experimentally. From the results of the activated dynamics simulations, local conformational .uctuations of the gorge residues and larger scale collective motions of the protein are found to correlate highly with the ligand crossing.

  16. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  17. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  18. Zinc Coordination Geometry and Ligand Binding Affinity: The Structural and Kinetic Analysis of the Second-Shell Serine 228 Residue and the Methionine 180 Residue of the Aminopeptidase from Vibrio proteolyticus

    SciTech Connect

    Ataie, Niloufar J.; Hoang, Quyen Q.; Zahniser, Megan P.D.; Tu, Yupeng; Milne, Amy; Petsko, Gregory A.; Ringe, Dagmar

    2008-07-28

    The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure?function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.

  19. Kinetic protein crystallography: a tool to watch proteins in action

    SciTech Connect

    Bourgeois, D.; Weik, M.

    2009-04-14

    Many proteins function in the crystalline state, making crystallography a tool that, beside structure, can address mechanism. By initiating biological turnover in the crystal, transient structural species form, which may be filmed 'on the fly' by Laue diffraction or captured by trapping methods. These strategies are jointly referred to as 'kinetic crystallography'. In this article, we review the general concepts of kinetic crystallography in the context of the conformational energy landscape of a protein. Whereas Laue diffraction is best suited to the investigation of cyclic, ultra-fast and light-triggered reactions, trapping approaches, on the other hand, are applicable to a wider range of biological systems but require care to avoid artefacts. Complementary methods - mainly UV/visible single-crystal spectroscopy - have proven essential to design, interpret and validate kinetic crystallography experiments. Achievements in the field as well as remaining puzzling questions are considered through the examination of recently published work: real-time-resolved crystallography of dimeric haemoglobin based on pump-probe Laue diffraction, temperature-trapping crystallography of acetylcholinesterase based on photo- and radio-induced ligand cleavage, and lattice-trapping crystallography of superoxide reductase based on product soaking and the combined use of X-ray diffraction and Raman spectroscopy.

  20. Ammonia formation by metal-ligand cooperative hydrogenolysis of a nitrido ligand

    NASA Astrophysics Data System (ADS)

    Askevold, Bjorn; Nieto, Jorge Torres; Tussupbayev, Samat; Diefenbach, Martin; Herdtweck, Eberhardt; Holthausen, Max C.; Schneider, Sven

    2011-07-01

    Bioinspired hydrogenation of N2 to ammonia at ambient conditions by stepwise nitrogen protonation/reduction with metal complexes in solution has experienced remarkable progress. In contrast, the highly desirable direct hydrogenation with H2 remains difficult. In analogy to the heterogeneously catalysed Haber-Bosch process, such a reaction is conceivable via metal-centred N2 splitting and unprecedented hydrogenolysis of the nitrido ligands to ammonia. We report the synthesis of a ruthenium(IV) nitrido complex. The high nucleophilicity of the nitrido ligand is demonstrated by unusual N-C coupling with π-acidic CO. Furthermore, the terminal nitrido ligand undergoes facile hydrogenolysis with H2 at ambient conditions to produce ammonia in high yield. Kinetic and quantum chemical examinations of this reaction suggest cooperative behaviour of a phosphorus-nitrogen-phosphorus pincer ligand in rate-determining heterolytic hydrogen splitting.

  1. Spectroscopic, Computational, and Kinetic Studies of the Mu-Sulfide-Bridged Tetranuclear CuZ Cluster in N(2)O Reductase: PH Effect on the Edge Ligand and its Contribution to Reactivity

    SciTech Connect

    Ghosh, S.; Gorelsky, S.I.; George, S.DeBeer; Chan, J.M.; Cabrito, I.; Dooley, D.M.; Moura, J.J.G.; Moura, I.; Solomon, E.I.

    2011-11-29

    A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the Cu{sub Z} site in Pseudomonas nautica (Pn) nitrous oxide reductase (N{sub 2}OR) and Achromobacter cycloclastes (Ac) N{sub 2}OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN{sub 2}OR at high pH shows that the 415 cm{sup -1} Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm{sup -1} {sup 18}O shift, implying significant Cu-O character, demonstrating the presence of a OH{sup -} ligand at the Cu{sub I}Cu{sub IV} edge. From DFT calculations, protonation of either the OH{sup -} to H{sub 2}O or the {mu}{sub 4}-S{sup 2-} to {mu}{sub 4}-SH{sup -} would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the Cu{sub I}Cu{sub IV} edge ligand show that the position of the OH{sup -} ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus, the observed pH effect (pK{sub a} {approx} 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E{sup o} and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu{sub 4}S(im){sub 7}OH]{sup 2+} to the fully reduced 4Cu{sup I} active form for turnover.

  2. Comparative DNA binding abilities and phosphatase-like activities of mono-, di-, and trinuclear Ni(II) complexes: the influence of ligand denticity, metal-metal distance, and coordinating solvent/anion on kinetics studies.

    PubMed

    Bhardwaj, Vimal K; Singh, Ajnesh

    2014-10-06

    Six novel Ni(II) complexes, namely, [Ni2(HL(1))(OAc)2] (1), [Ni3L(1)2]·H2O·2CH3CN (2), [Ni2(L(2))(L(3))(CH3CN)] (3), [Ni2(L(2))2(H2O)2] (4), [Ni2(L(2))2(DMF)2]2·2H2O (5), and [Ni(HL(2))2]·H2O (6), were synthesized by reacting nitrophenol-based tripodal (H3L(1)) and dipodal (H2L(2)) Schiff base ligands with Ni(II) metal salts at ambient conditions. All the complexes were fully characterized with different spectroscopic techniques such as elemental analyses, IR, UV-vis spectroscopy, and electrospray ionization mass spectrometry. The solid-state structures of 2, 3, 5, and 6 were determined using single-crystal X-ray crystallography. The compounds 1, 3, 4, and 5 are dinuclear complexes where the two Ni(II) centers have octahedral geometry with bridging phenoxo groups. Compound 2 is a trinuclear complex with two different types of Ni(II) centers. In compound 3 one of the Ni(II) centers has a coordinated acetonitrile molecule, whereas in compound 4, a water molecule has occupied one coordination site of each Ni(II) center. In complex 5, the coordinated water of complex 4 was displaced by the dimethylformamide (DMF) during its crystallization. Complex 6 is mononuclear with two amine-bis(phenolate) ligands in scissorlike fashion around the Ni(II) metal center. The single crystals of 1 and 4 could not be obtained; however, from the spectroscopic data and physicochemical properties (electronic and redox properties) it was assumed that the structures of these complexes are quite similar to other analogues. DNA binding abilities and phosphatase-like activities of all characterized complexes were also investigated. The ligand denticity, coordinated anions/solvents (such as acetate, acetonitrile, water, and DMF), and cooperative action of two metal centers play a significant role in the phosphate ester bond cleavage of 2-hydroxypropyl-p-nitropenylphosphate by transesterification mechanism. Complex 3 exhibits highest activity among complexes 1-6 with 3.86 × 10(5) times

  3. Gating Kinetics of the Cyclic-GMP-Activated Channel of Retinal Rods: Flash Photolysis and Voltage-Jump Studies

    NASA Astrophysics Data System (ADS)

    Karpen, Jeffrey W.; Zimmerman, Anita L.; Stryer, Lubert; Baylor, Denis A.

    1988-02-01

    The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester (``caged'' cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (>50 μ M), whereas at physiological cGMP concentrations (<5 μ M), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.

  4. Fast Reactors

    NASA Astrophysics Data System (ADS)

    Esposito, S.; Pisanti, O.

    The following sections are included: * Elementary Considerations * The Integral Equation to the Neutron Distribution * The Critical Size for a Fast Reactor * Supercritical Reactors * Problems and Exercises

  5. Ruthenium-based olefin metathesis catalysts bearing pH-responsive ligands: External control of catalyst solubility and activity

    NASA Astrophysics Data System (ADS)

    Balof, Shawna Lynn

    2011-12-01

    Sixteen novel, Ru-based olefin metathesis catalysts bearing pH responsive ligands were synthesized. The pH-responsive groups employed with these catalysts included dimethylamino (NMe2) modified NHC ligands as well as N-donor dimethylaminopyridine (DMAP) and 3-(o-pyridyl)propylidene ligands. These pH-responsive ligands provided the means by which the solubility and/or activity profiles of the catalysts produced could be controlled via acid addition. The main goal of this dissertation was to design catalyst systems capable of performing ring opening metathesis (ROMP) and ring closing metathesis (RCM) reactions in both organic and aqueous media. In an effort to quickly gain access to new catalyst structures, a template synthesis for functionalized NHC ligand precursors was designed, in addition to other strategies, to obtain ligand precursors with ancillary NMe2 groups. Kinetic studies for the catalysts produced from these precursors showed external control of catalyst solubility was afforded via protonation of the NMe2 groups of their NHC ligands. Additionally, this protonation afforded external control of catalyst propagation rates for several catalysts. This is the first known independent external control for the propagation rates of ROMP catalysts. The incorporation of pH-responsive N-donor ligands into catalyst structures also provided the means for the external control of metathesis activity, as the protonation of these ligands resulted in an increased initiation rate based on their fast and irreversible dissociation from the metal center. The enhanced external control makes these catalysts applicable to a wide range of applications, some of which have been explored by us and/or through collaboration. Three of the catalysts designed showed remarkable metathesis activity in aqueous media. These catalysts displayed comparable RCM activity in aqueous media to a class of water-soluble catalysts reported by Grubbs et al., considered to be the most active catalyst for

  6. Water network perturbation in ligand binding: adenosine A(2A) antagonists as a case study.

    PubMed

    Bortolato, Andrea; Tehan, Ben G; Bodnarchuk, Michael S; Essex, Jonathan W; Mason, Jonathan S

    2013-07-22

    Recent efforts in the computational evaluation of the thermodynamic properties of water molecules have resulted in the development of promising new in silico methods to evaluate the role of water in ligand binding. These methods include WaterMap, SZMAP, GRID/CRY probe, and Grand Canonical Monte Carlo simulations. They allow the prediction of the position and relative free energy of the water molecule in the protein active site and the analysis of the perturbation of an explicit water network (WNP) as a consequence of ligand binding. We have for the first time extended these approaches toward the prediction of kinetics for small molecules and of relative free energy of binding with a focus on the perturbation of the water network and application to large diverse data sets. Our results support a qualitative correlation between the residence time of 12 related triazine adenosine A(2A) receptor antagonists and the number and position of high energy trapped solvent molecules. From a quantitative viewpoint, we successfully applied these computational techniques as an implicit solvent alternative, in linear combination with a molecular mechanics force field, to predict the relative ligand free energy of binding (WNP-MMSA). The applicability of this linear method, based on the thermodynamics additivity principle, did not extend to 375 diverse A(2A) receptor antagonists. However, a fast but effective method could be enabled by replacing the linear approach with a machine learning technique using probabilistic classification trees, which classified the binding affinity correctly for 90% of the ligands in the training set and 67% in the test set.

  7. Kinetic regulation mechanism of pbuE riboswitch

    NASA Astrophysics Data System (ADS)

    Gong, Sha; Wang, Yujie; Zhang, Wenbing

    2015-01-01

    Riboswitches are RNA residue segments located in untranslated regions of messenger RNAs. These folded segments directly bind ligands through shape complementarity and specific interactions in cells and alter the expression of genes at the transcriptional or translational level through conformation change. Using the recently developed systematic helix-based computational method to predict the cotranscription folding kinetics, we theoretically studied the cotranscription folding behavior of the Bacillus subtilis pbuE riboswitch in the absence and presence of the ligand. The ligand concentration, the transcription speed, and the transcription pausing are incorporated into the method. The results are in good agreement with the experimental results. We find that there are no obvious misfolded structures formed during the transcription and the formation of the ligand bound state is rate-limited by the association of the ligand and the RNA. For this kinetically driven riboswitch, the ligand concentration, the transcription speed, and the transcription pausing are coupled to perform regulatory activity.

  8. Hysteresis of ligand binding in CNGA2 ion channels

    PubMed Central

    Nache, Vasilica; Eick, Thomas; Schulz, Eckhard; Schmauder, Ralf; Benndorf, Klaus

    2013-01-01

    Tetrameric cyclic nucleotide-gated (CNG) channels mediate receptor potentials in olfaction and vision. The channels are activated by the binding of cyclic nucleotides to a binding domain embedded in the C terminus of each subunit. Here using a fluorescent cGMP derivative (fcGMP), we show for homotetrameric CNGA2 channels that ligand unbinding is ~50 times faster at saturating than at subsaturating fcGMP. Analysis with complex Markovian models reveals two pathways for ligand unbinding; the partially liganded open channel unbinds its ligands from closed states only, whereas the fully liganded channel reaches a different open state from which it unbinds all four ligands rapidly. Consequently, the transition pathways for ligand binding and activation of a fully liganded CNGA2 channel differ from that of ligand unbinding and deactivation, resulting in pronounced hysteresis of the gating mechanism. This concentration-dependent gating mechanism allows the channels to respond to changes in the cyclic nucleotide concentration with different kinetics. PMID:24287615

  9. Group IV organometallic compounds based on dianionic "pincer" ligands: synthesis, characterization, and catalytic activity in intramolecular hydroamination reactions.

    PubMed

    Luconi, Lapo; Rossin, Andrea; Motta, Alessandro; Tuci, Giulia; Giambastiani, Giuliano

    2013-04-08

    Neutral Zr(IV) and Hf(IV) diamido complexes stabilized by unsymmetrical dianionic N,C,N' pincer ligands have been prepared through the simplest and convenient direct metal-induced Caryl-H bond activation. Simple ligand modification has contributed to highlight the non-innocent role played by the donor atom set in the control of the cyclometallation kinetics. The as-prepared bis-amido catalysts were found to be good candidates for the intramolecular hydroamination/cyclization of primary aminoalkenes. The ability of these compounds to promote such a catalytic transformation efficiently (by providing, in some cases, fast and complete substrate conversion at room temperature) constitutes a remarkable step forward toward catalytic systems that can operate at relatively low catalyst loading and under milder reaction conditions. Kinetic studies and substrate-scope investigations, in conjunction with preliminary DFT calculations on the real systems, were used to elucidate the effects of the substrate substitution on the catalyst performance and to support the most reliable mechanistic path operative in the hydroamination reaction.

  10. Kinetic study on biomass gasification

    SciTech Connect

    Bingyan, X.; Chuangzhi, W.; Zhengfen, L.; Guang, Z.X. )

    1992-09-01

    An experimental apparatus, with the features of fast heating rate and continuous record of reaction parameters, was developed to study kinetics of fast pyrolysis. The temperature effects, at a range of 400 C to 900 C, on pyrolysis rate, products profile, gas quality and quantity, and so on, were studied and the results are listed and analyzed. The effect of secondary reaction of gas phase at 700 C was tested and the regression result is expressed in an experimental formula. Based on the experimental results, the three-stage-reaction mechanism module is suggested. The kinetic expression to calculate gas formation rate is concluded as: d{alpha}/dt = A exp({minus}E/RT)(1 {minus} {alpha}){sup n}. The kinetic parameters of A, E, and n at different temperatures are given in the paper.

  11. Broad-spectrum kinetic resolution of alcohols enabled by Cu-H-catalysed dehydrogenative coupling with hydrosilanes

    NASA Astrophysics Data System (ADS)

    Dong, Xichang; Weickgenannt, Andreas; Oestreich, Martin

    2017-06-01

    The enantioselective silylation of racemic alcohols, where one enantiomer reacts faster than the other, is an alternative approach to established enzymatic and non-enzymatic acylation techniques. The existing art is either limited to structurally biased alcohols or requires elaborate catalysts. Simple substrates, such as benzylic and allylic alcohols, with no coordinating functionality in the proximity of the hydroxy group have been challenging in these kinetic resolutions. We report here the identification of a broadly applicable chiral catalyst for the enantioselective dehydrogenative coupling of alcohols and hydrosilanes with both the chiral ligand and the hydrosilane being commercially available. The efficiency of kinetic resolutions is characterized by the selectivity factor, that is, the ratio of the reaction rates of the fast-reacting over the slow-reacting enantiomer. The selectivity factors achieved with the new method are good for acyclic benzylic alcohols (<=170) and high for synthetically usefully cyclic benzylic (<=40.1) and allylic alcohols (<=159).

  12. Ligand modeling and design

    SciTech Connect

    Hay, B.P.

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  13. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation

    PubMed Central

    Moriarty, Nigel W.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.

    2009-01-01

    The electronic Ligand Builder and Optimization Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallographic software. It is designed to be a flexible procedure that uses simple and fast quantum-chemical techniques to provide chemically accurate information for novel and known ligands alike. A variety of input formats and options allow the attainment of a number of diverse goals including geometry optimization and generation of restraints. PMID:19770504

  14. electronic Ligand Builder and Optimisation Workbench (eLBOW): A tool for ligand coordinate and restraint generation

    SciTech Connect

    Moriarty, Nigel; Grosse-Kunstleve, Ralf; Adams, Paul

    2009-07-01

    The electronic Ligand Builder and Optimisation Workbench (eLBOW) is a program module of the PHENIX suite of computational crystallographic software. It's designed to be a flexible procedure using simple and fast quantum chemical techniques to provide chemically accurate information for novel and known ligands alike. A variety of input formats and options allow for the attainment of a number of diverse goals including geometry optimisation and generation of restraints.

  15. Diffusion modifies the connectivity of kinetic schemes for multisite binding and catalysis

    PubMed Central

    Gopich, Irina V.; Szabo, Attila

    2013-01-01

    The simplest way to describe the influence of the relative diffusion of the reactants on the time course of bimolecular reactions is to modify or renormalize the phenomenological rate constants that enter into the rate equations of conventional chemical kinetics. However, for macromolecules with multiple inequivalent reactive sites, this is no longer sufficient, even in the low concentration limit. The physical reason is that an enzyme (or a ligand) that has just modified (or dissociated from) one site can bind to a neighboring site rather than diffuse away. This process is not described by the conventional chemical kinetics, which is only valid in the limit that diffusion is fast compared with reaction. Using an exactly solvable many-particle reaction-diffusion model, we show that the influence of diffusion on the kinetics of multisite binding and catalysis can be accounted for by not only scaling the rates, but also by introducing new connections into the kinetic scheme. The rate constants that describe these new transitions or reaction channels turn out to have a transparent physical interpretation: The chemical rates are scaled by the appropriate probabilities that a pair of reactants, which are initially in contact, bind rather than diffuse apart. The theory is illustrated by application to phosphorylation of a multisite substrate. PMID:24248348

  16. Diffusion modifies the connectivity of kinetic schemes for multisite binding and catalysis.

    PubMed

    Gopich, Irina V; Szabo, Attila

    2013-12-03

    The simplest way to describe the influence of the relative diffusion of the reactants on the time course of bimolecular reactions is to modify or renormalize the phenomenological rate constants that enter into the rate equations of conventional chemical kinetics. However, for macromolecules with multiple inequivalent reactive sites, this is no longer sufficient, even in the low concentration limit. The physical reason is that an enzyme (or a ligand) that has just modified (or dissociated from) one site can bind to a neighboring site rather than diffuse away. This process is not described by the conventional chemical kinetics, which is only valid in the limit that diffusion is fast compared with reaction. Using an exactly solvable many-particle reaction-diffusion model, we show that the influence of diffusion on the kinetics of multisite binding and catalysis can be accounted for by not only scaling the rates, but also by introducing new connections into the kinetic scheme. The rate constants that describe these new transitions or reaction channels turn out to have a transparent physical interpretation: The chemical rates are scaled by the appropriate probabilities that a pair of reactants, which are initially in contact, bind rather than diffuse apart. The theory is illustrated by application to phosphorylation of a multisite substrate.

  17. Non-Michaelis-Menten kinetics in cytochrome P450-catalyzed reactions.

    PubMed

    Atkins, William M

    2005-01-01

    The cytochrome P450 monooxygenases (CYPs) are the dominant enzyme system responsible for xenobiotic detoxification and drug metabolism. Several CYP isoforms exhibit non-Michaelis-Menten, or "atypical," steady state kinetic patterns. The allosteric kinetics confound prediction of drug metabolism and drug-drug interactions, and they challenge the theoretical paradigms of allosterism. Both homotropic and heterotropic ligand effects are now widely documented. It is becoming apparent that multiple ligands can simultaneously bind within the active sites of individual CYPs, and the kinetic parameters change with ligand occupancy. In fact, the functional effect of any specific ligand as an activator or inhibitor can be substrate dependent. Divergent approaches, including kinetic modeling and X-ray crystallography, are providing new information about how multiple ligand binding yields complex CYP kinetics.

  18. Equilibrium and kinetic studies on complex formation and decomposition and the movement of Cu(2+)metal ions within polytopic receptors.

    PubMed

    Castillo, Carmen Ester; González-García, Jorge; Llinares, José M; Máñez, M Angeles; Jimenez, Hermas R; García-España, Enrique; Basallote, Manuel G

    2013-05-07

    Potentiometric studies carried out on the interaction of two tritopic double-scorpiand receptors in which two equivalent 5-(2-aminoethyl)-2,5,8-triaza[9]-(2,6)-pyridinophane moieties are linked with 2,9-dimethylphenanthroline (L1) and 2,6-dimethylpyridine (L2) establish the formation of mono-, bi- and trinuclear Cu(2+) complexes. The values of the stability constants and paramagnetic (1)H NMR studies permit one to infer the most likely coordination modes of the various complexes formed. Kinetic studies on complex formation and decomposition have also been carried out. Complex formation occurs with polyphasic kinetics for both receptors, although a significant difference is found between both ligands with respect to the relative values of the rate constants for the metal coordination steps and the structural reorganizations following them. Complex decomposition occurs with two separate kinetic steps, the first one being so fast that it occurs within the stopped-flow mixing time, whereas the second one is slow enough to allow kinetic studies using a conventional spectrophotometer. As a whole, the kinetic experiments also provide information about the movement of the metal ion within the receptors. The differences observed between the different receptors can be interpreted in terms of changes in the network of hydrogen bonds formed in the different species.

  19. Ligand modeling and design

    SciTech Connect

    Hay, B.

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  20. [Kinetics of conformational changes of methemoglobin complexed with liposomes].

    PubMed

    Gorbenko, G P

    1998-01-01

    Kinetics of methemoglobin structural changes in the complex with liposomes composed of phosphatidylcholine and its mixtures with cardiolipin has been studied. The amplitudes and rate constants of the two observed kinetic phases are determined. The fast kinetic phase is attributed to the formation of the unstable intermediate protein form, while the slow one is assumed to reflect dissociation of the heme--globin complex.

  1. Kinetics of excitation in TL and OSL detectors

    NASA Astrophysics Data System (ADS)

    Mandowski, A.; Orzechowski, J.; Mandowska, E.

    2010-10-01

    Kinetic equations for the semi-localized transitions (SLT) model are presented describing charge carrier's kinetics for the excitation and fast relaxation stages. The formulation allows for dose dependence studies of thermoluminescence (TL) and optically stimulated luminescence (OSL) detectors based on the SLT model. The sets of equations were solved numerically demonstrating temporal evolution of all variables of the SLT model during excitation and fast relaxation. The influence of the dose rate on excitation kinetics is shown.

  2. Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

    PubMed

    Lill, Yoriko; Martinez, Karen L; Lill, Markus A; Meyer, Bruno H; Vogel, Horst; Hecht, Bert

    2005-08-12

    We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.

  3. Quantum.Ligand.Dock: protein–ligand docking with quantum entanglement refinement on a GPU system

    PubMed Central

    Kantardjiev, Alexander A.

    2012-01-01

    Quantum.Ligand.Dock (protein–ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein–ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments—the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein–ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pKa values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html. PMID:22669908

  4. Quantum.Ligand.Dock: protein-ligand docking with quantum entanglement refinement on a GPU system.

    PubMed

    Kantardjiev, Alexander A

    2012-07-01

    Quantum.Ligand.Dock (protein-ligand docking with graphic processing unit (GPU) quantum entanglement refinement on a GPU system) is an original modern method for in silico prediction of protein-ligand interactions via high-performance docking code. The main flavour of our approach is a combination of fast search with a special account for overlooked physical interactions. On the one hand, we take care of self-consistency and proton equilibria mutual effects of docking partners. On the other hand, Quantum.Ligand.Dock is the the only docking server offering such a subtle supplement to protein docking algorithms as quantum entanglement contributions. The motivation for development and proposition of the method to the community hinges upon two arguments-the fundamental importance of quantum entanglement contribution in molecular interaction and the realistic possibility to implement it by the availability of supercomputing power. The implementation of sophisticated quantum methods is made possible by parallelization at several bottlenecks on a GPU supercomputer. The high-performance implementation will be of use for large-scale virtual screening projects, structural bioinformatics, systems biology and fundamental research in understanding protein-ligand recognition. The design of the interface is focused on feasibility and ease of use. Protein and ligand molecule structures are supposed to be submitted as atomic coordinate files in PDB format. A customization section is offered for addition of user-specified charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. Final predicted complexes are ranked according to obtained scores and provided in PDB format as well as interactive visualization in a molecular viewer. Quantum.Ligand.Dock server can be accessed at http://87.116.85.141/LigandDock.html.

  5. New approach to a novel axially chiral ligand showing spontaneous enrichment of axial chirality.

    PubMed

    Hasegawa, Tomokuni; Omote, Masaaki; Sato, Kazuyuki; Ando, Akira; Kumadaki, Itsumaro

    2003-03-01

    We have synthesized novel axially chiral ligand with two chiral centers, (R)-(R)(2)- and (S)-(S)(2)-2,2'-bis(2,2,2-trifluoro-1-hydroxyethyl)biphenyl (1), which showed a high asymmetric induction when used as ligand. Here, another new approach to 1 by kinetic and thermodynamic resolution is presented which gave these ligands in a much shorter steps, in a higher yield, and in a higher enantiomeric excess.

  6. Fast valve

    SciTech Connect

    Van Dyke, William J.

    1992-01-01

    A fast valve is disclosed that can close on the order of 7 milliseconds. It is closed by the force of a compressed air spring with the moving parts of the valve designed to be of very light weight and the valve gate being of wedge shaped with O-ring sealed faces to provide sealing contact without metal to metal contact. The combination of the O-ring seal and an air cushion create a soft final movement of the valve closure to prevent the fast air acting valve from having a harsh closing.

  7. Fast valve

    DOEpatents

    Van Dyke, W.J.

    1992-04-07

    A fast valve is disclosed that can close on the order of 7 milliseconds. It is closed by the force of a compressed air spring with the moving parts of the valve designed to be of very light weight and the valve gate being of wedge shaped with O-ring sealed faces to provide sealing contact without metal to metal contact. The combination of the O-ring seal and an air cushion create a soft final movement of the valve closure to prevent the fast air acting valve from having a harsh closing. 4 figs.

  8. Determining enzyme kinetics via isothermal titration calorimetry.

    PubMed

    Demarse, Neil A; Killian, Marie C; Hansen, Lee D; Quinn, Colette F

    2013-01-01

    Isothermal titration calorimetry (ITC) has emerged as a powerful tool for determining the thermodynamic properties of chemical or physical equilibria such as protein-protein, ligand-receptor, and protein-DNA binding interactions. The utility of ITC for determining kinetic information, however, has not been fully recognized. Methods for collecting and analyzing data on enzyme kinetics are discussed here. The step-by-step process of converting the raw heat output rate into the kinetic parameters of the Michaelis-Menten equation is explicitly stated. The hydrolysis of sucrose by invertase is used to demonstrate the capability of the instrument and method.

  9. Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

    PubMed

    Segala, Elena; Guo, Dong; Cheng, Robert K Y; Bortolato, Andrea; Deflorian, Francesca; Doré, Andrew S; Errey, James C; Heitman, Laura H; IJzerman, Adriaan P; Marshall, Fiona H; Cooke, Robert M

    2016-07-14

    The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

  10. Complications in complexation kinetics for lanthanides with DTPA using dye probe molecules in aqueous solution

    DOE PAGES

    Larsson, K.; Cullen, T. D.; Mezyk, S. P.; ...

    2017-05-17

    The complexation kinetics for the polyaminopolycarboxylic ligand DTPA to lanthanides in acidic aqueous solution were investigated using the dye ligand displacement technique and stopped-flow spectroscopy. Significant rate differences were obtained for different dye probes used, indicating that the kinetics of the dissociation of the dye molecule significantly impacts the overall measured kinetics when using this common methodology. The conditions of the solution also influenced the dye-lanthanide-DTPA interactions, which reconciled previously disparate data in the literature.

  11. Kinetic titration series with biolayer interferometry.

    PubMed

    Frenzel, Daniel; Willbold, Dieter

    2014-01-01

    Biolayer interferometry is a method to analyze protein interactions in real-time. In this study, we illustrate the usefulness to quantitatively analyze high affinity protein ligand interactions employing a kinetic titration series for characterizing the interactions between two pairs of interaction patterns, in particular immunoglobulin G and protein G B1 as well as scFv IC16 and amyloid beta (1-42). Kinetic titration series are commonly used in surface plasmon resonance and involve sequential injections of analyte over a desired concentration range on a single ligand coated sensor chip without waiting for complete dissociation between the injections. We show that applying this method to biolayer interferometry is straightforward and i) circumvents problems in data evaluation caused by unavoidable sensor differences, ii) saves resources and iii) increases throughput if screening a multitude of different analyte/ligand combinations.

  12. Kinetic Titration Series with Biolayer Interferometry

    PubMed Central

    Frenzel, Daniel; Willbold, Dieter

    2014-01-01

    Biolayer interferometry is a method to analyze protein interactions in real-time. In this study, we illustrate the usefulness to quantitatively analyze high affinity protein ligand interactions employing a kinetic titration series for characterizing the interactions between two pairs of interaction patterns, in particular immunoglobulin G and protein G B1 as well as scFv IC16 and amyloid beta (1–42). Kinetic titration series are commonly used in surface plasmon resonance and involve sequential injections of analyte over a desired concentration range on a single ligand coated sensor chip without waiting for complete dissociation between the injections. We show that applying this method to biolayer interferometry is straightforward and i) circumvents problems in data evaluation caused by unavoidable sensor differences, ii) saves resources and iii) increases throughput if screening a multitude of different analyte/ligand combinations. PMID:25229647

  13. Project FAST.

    ERIC Educational Resources Information Center

    Essexville-Hampton Public Schools, MI.

    Described are components of Project FAST (Functional Analysis Systems Training) a nationally validated project to provide more effective educational and support services to learning disordered children and their regular elementary classroom teachers. The program is seen to be based on a series of modules of delivery systems ranging from mainstream…

  14. Fast CRCs

    DTIC Science & Technology

    2009-10-01

    or any other computing topic, please visit our Digital Library at www.computer.org/publications/ dlib . NGUYEN: FAST CRCS 1331 Authorized licensed use... Library at http://doi.ieeecomputersociety.org/10.1109/ TC.2009.83. The notation ðk; l; dÞ denotes a systematic code with k ¼ the total bit length of

  15. Lipid metabolism during fasting.

    PubMed

    Jensen, M D; Ekberg, K; Landau, B R

    2001-10-01

    These studies were conducted to understand the relationship between measures of systemic free fatty acid (FFA) reesterification and regional FFA, glycerol, and triglyceride metabolism during fasting. Indirect calorimetry was used to measure fatty acid oxidation in six men after a 60-h fast. Systemic and regional (splanchnic, renal, and leg) FFA ([(3)H]palmitate) and glycerol ([(3)H]glycerol) kinetics, as well as splanchnic triglyceride release, were measured. The rate of systemic FFA reesterification was 366 +/- 93 micromol/min, which was greater (P < 0.05) than splanchnic triglyceride fatty acid output (64 +/- 6 micromol/min), a measure of VLDL triglyceride fatty acid export. The majority of glycerol uptake occurred in the splanchnic and renal beds, although some leg glycerol uptake was detected. Systemic FFA release was approximately double that usually present in overnight postabsorptive men, yet the regional FFA release rates were of the same proportions previously observed in overnight postabsorptive men. In conclusion, FFA reesterification at rest during fasting far exceeds splanchnic triglyceride fatty acid output. This indicates that nonhepatic sites of FFA reesterification are important, and that peripheral reesterification of FFA exceeds the rate of simultaneous intracellular triglyceride fatty acid oxidation.

  16. Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

    PubMed Central

    Camunas-Soler, Joan; Manosas, Maria; Frutos, Silvia; Tulla-Puche, Judit; Albericio, Fernando; Ritort, Felix

    2015-01-01

    DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines. PMID:25690887

  17. Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling.

    PubMed

    Van Belle, D; De Maria, L; Iurcu, G; Wodak, S J

    2000-05-12

    somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release. Copyright 2000 Academic Press.

  18. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  19. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  20. Revealing equilibrium and rate constants of weak and fast noncovalent interactions.

    PubMed

    Mironov, Gleb G; Okhonin, Victor; Gorelsky, Serge I; Berezovski, Maxim V

    2011-03-15

    Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 μM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K(d) > 80 μM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and β-cyclodextrins. The affinity of the drugs was significantly higher for β-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation.

  1. Further Insight into the Lability of MeCN Ligands of Cytotoxic Cycloruthenated Compounds: Evidence for the Antisymbiotic Effect Trans to the Carbon Atom at the Ru Center.

    PubMed

    Barbosa, Ana Soraya Lima; Werlé, Christophe; Colunga, Claudia Olivia Oliva; Rodríguez, Cecilia Franco; Toscano, Ruben Alfredo; Le Lagadec, Ronan; Pfeffer, Michel

    2015-08-03

    The two MeCN ligands in [Ru(2-C6H4-2'-Py-κC,N)(Phen, trans-C)(MeCN)2]PF6 (1), both trans to a sp(2) hybridized N atom, cannot be substituted by any other ligand. In contrast, the isomerized derivative [Ru(2-C6H4-2'-Py-κC,N)(Phen, cis-C)(MeCN)2]PF6 (2), in which one MeCN ligand is now trans to the C atom of the phenyl ring orthometalated to Ru, leads to fast and quantitative substitution reactions with several monodentate ligands. With PPh3, 2 affords [Ru(2-C6H4-2'-Py-κC,N)(Phen, cis-C)(PPh3)(MeCN)]PF6 (3), in which PPh3 is trans to the C σ bound to Ru. Compound 3 is not kinetically stable, because, under thermodynamic control, it leads to 4, in which the PPh3 is trans to a N atom of the Phen ligand. Dimethylsulfoxide (DMSO) can also substitute a MeCN ligand in 2, leading to 5, in which DMSO is coordinated to Ru via its S atom trans to the N atom of the Phen ligand, the isomer under thermodynamic control being the only compound observed. We also found evidence for the fast to very fast substitution of MeCN in 2 by water or a chloride anion by studying the electronic spectra of 2 in the presence of water or NBu4Cl, respectively. An isomerization related to that observed between 3 and 4 is also found for the known monophosphine derivative [Ru(2-C6H4-2'-Py-κC,N)(PPh3, trans-C)(MeCN)3]PF6 (10), in which the PPh3 is located trans to the C of the cyclometalated 2-phenylpyridine, since, upon treatment by refluxing MeCN, it leads to its isomer 11, [Ru(2-C6H4-2'-Py-κC,N)(PPh3, cis-C)(MeCN)3]PF6. Further substitutions are also observed on 11, whereby N^N chelates (N^N = 2,2'-bipyridine and phenanthroline) substitute two MeCN ligands, affording [Ru(2-C6H4-2'-Py-κC,N)(PPh3, cis-C)(N^N)(MeCN)]PF6 (12a and 12b). Altogether, the behavior of the obtained complexes by ligand substitution reactions can be rationalized by an antisymbiotic effect on the Ru center, trans to the C atom of the cyclometalated unit, leading to compounds having the least nucleophilic ligand trans to C

  2. On the significance of the retention of ligand by protein.

    PubMed Central

    Silhavy, T J; Szmelcman, S; Boos, W; Schwartz, M

    1975-01-01

    When a solution of binding protein and its ligand is dialyzed against a large volume of ligand-free medium the rate of exit of the ligand from the protein-containing compartment can be extremely slow, much slower than the rate observed in the absence of protein. This is what we call retention of ligand by protein. A simple calculation demonstrates that when the protein concentration is in large excess over the total ligand concentration, the exit of ligand follows quasi-first-order kinetics, the half-life being proportional to (1 + (P)/Kd), where (P) is the concentration of binding sites, and Kd the dissociation constant characteristic of the equilibrium between the ligand and the protein. Experimental verification of this relation is provided in the case of the periplasmic maltose-binding protein of Escherichia coli; The implications of the retention effect in biochemical techniques are discussed, as well as its possible significance in biological phenomena, such as bacterial chemotaxis and transport, mechanism of hormone action, or transmission of the nerve impulse. PMID:1094454

  3. Effect of surface ligands on the optical properties of aqueous soluble CdTe quantum dots

    PubMed Central

    2012-01-01

    We investigate systematically the influence of the nature of thiol-type capping ligands on the optical and structural properties of highly luminescent CdTe quantum dots synthesized in aqueous media, comparing mercaptopropionic acid (MPA), thioglycolic acid (TGA), 1-thioglycerol (TGH), and glutathione (GSH). The growth rate, size distribution, and quantum yield strongly depend on the type of surface ligand used. While TGH binds too strongly to the nanocrystal surface inhibiting growth, the use of GSH results in the fastest growth kinetics. TGA and MPA show intermediate growth kinetics, but MPA yields a much lower initial size distribution than TGA. The obtained fluorescence quantum yields range from 38% to 73%. XPS studies unambiguously put into evidence the formation of a CdS shell on the CdTe core due to the thermal decomposition of the capping ligands. This shell is thicker when GSH is used as ligand, as compared with TGA ligands. PMID:23017183

  4. Ligand fitting with CCP4

    PubMed Central

    2017-01-01

    Crystal structures of protein–ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein–ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation. PMID:28177312

  5. Kinetic percolation

    NASA Astrophysics Data System (ADS)

    Heinson, W. R.; Chakrabarti, A.; Sorensen, C. M.

    2017-05-01

    We demonstrate that kinetic aggregation forms superaggregates that have structures identical to static percolation aggregates, and these superaggregates appear as a separate phase in the size distribution. Diffusion limited cluster-cluster aggregation (DLCA) simulations were performed to yield fractal aggregates with a fractal dimension of 1.8 and superaggregates with a fractal dimension of D = 2.5 composed of these DLCA supermonomers. When properly normalized to account for the DLCA fractal nature of their supermonomers, these superaggregates have the exact same monomer packing fraction, scaling law prefactor, and scaling law exponent (the fractal dimension) as percolation aggregates; these are necessary and sufficient conditions for same structure. The size distribution remains monomodal until these superaggregates form to alter the distribution. Thus the static percolation and the kinetic descriptions of gelation are now unified.

  6. Balancing specificity, sensitivity, and speed of ligand discrimination by zero-order ultraspecificity

    NASA Astrophysics Data System (ADS)

    Kajita, Masashi K.; Aihara, Kazuyuki; Kobayashi, Tetsuya J.

    2017-07-01

    Specific interactions between receptors and their target ligands in the presence of nontarget ligands are crucial for biological processes such as T cell ligand discrimination. To discriminate between the target and nontarget ligands, cells have to increase specificity to the target ligands by amplifying the small differences in affinity among ligands. In addition, sensitivity to the ligand concentration and quick discrimination are also important to detect low amounts of target ligands and facilitate fast cellular decision making after ligand recognition. In this work we propose a mechanism for nonlinear specificity amplification (ultraspecificity) based on zero-order saturating reactions, which was originally proposed to explain nonlinear sensitivity amplification (ultrasensitivity) to the ligand concentration. In contrast to the previously proposed proofreading mechanisms that amplify the specificity by a multistep reaction, our model can produce an optimal balance of specificity, sensitivity, and quick discrimination. Furthermore, we show that a model for insensitivity to a large number of nontarget ligands can be naturally derived from a model with the zero-order ultraspecificity. The zero-order ultraspecificity, therefore, may provide an alternative way to understand ligand discrimination from the viewpoint of nonlinear properties in biochemical reactions.

  7. Ligand efficiency metrics considered harmful

    NASA Astrophysics Data System (ADS)

    Kenny, Peter W.; Leitão, Andrei; Montanari, Carlos A.

    2014-07-01

    Ligand efficiency metrics are used in drug discovery to normalize biological activity or affinity with respect to physicochemical properties such as lipophilicity and molecular size. This Perspective provides an overview of ligand efficiency metrics and summarizes thermodynamics of protein-ligand binding. Different classes of ligand efficiency metric are critically examined and the study concludes with suggestions for alternative ways to account for physicochemical properties when prioritizing and optimizing leads.

  8. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

    Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

  9. Investigations of ultrafast ligand rebinding to heme and heme proteins using temperature and strong magnetic field perturbations

    NASA Astrophysics Data System (ADS)

    Zhang, Zhenyu

    study the ligand recombination after photolysis. No magnetic field induced rate changes are observed in any of these ligand recombination processes within the experimental detection limit. A magnetic field dependent CO rebinding behavior is observed for the FePPIX-CO sample in 80%glycerol/20%water environment. Careful data analysis indicates that this magnetic field induced change is due to the amplitude difference of a "fast" (<10ps) response with and without the magnetic field application (the amplitude changes from ˜55% at 0 Tesla to ˜45% at 10 Tesla). Kinetics of CO rebinding to FePPIX in 80%glycerol at the extremes of the magnetic field intensities (0Tesla vs. 10 Tesla) can be decomposed into a ligand rebinding process plus two 5ps decays heme cooling with different amplitudes. It leads to suggest a magnetic field induced change of a short-lived heme cooling response after photolysis. Also, CO rebinding kinetics to different heme compounds demonstrates a wide range for the Arrhenius pre-factors. This work reveals that the "spin-selection rule" does not play a key role in the recombination process of CO to heme iron. In Appendix 1, the recombination of oxymyoglobin and its mutants is investigated in the temperature range from 275K to 318K, using a home-made cryostat. Quite surprisingly, the O2 molecule rebinds to heme iron inside myoglobin with dramatically different behavior as the temperature is varied, depending on the protein environment. It shows little dependence (Mb), no dependence (V68W Mb mutant) and large dependence (L29W Mb mutant) in this 40K temperature window. To expand this temperature window, since the motor inside the cryostat is capable to work as low as 230K, glycerol is introduced into the protein preparation. It is observed that protein samples in a glycerol/water mixture, even with only 20% glycerol (in weight), the temperature dependences of the O2 rebinding to heme iron are dramatically altered. The O 2 rebinding behavior also shows a high

  10. LigandRNA: computational predictor of RNA-ligand interactions.

    PubMed

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  11. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    DOEpatents

    Von Dreele, Robert B [Los Alamos, NM

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  12. Complexation of metal ions with TRAP (1,4,7-triazacyclononane phosphinic acid) ligands and 1,4,7-triazacyclononane-1,4,7-triacetic acid: phosphinate-containing ligands as unique chelators for trivalent gallium.

    PubMed

    Šimeček, Jakub; Schulz, Martin; Notni, Johannes; Plutnar, Jan; Kubíček, Vojtěch; Havlíčková, Jana; Hermann, Petr

    2012-01-02

    Three phosphinic acid 1,4,7-triazacyclononane (TACN) derivatives bearing methylphosphinic (TRAP-H), methyl(phenyl)phosphinic (TRAP-Ph), or methyl(hydroxymethyl)phosphinic acid (TRAP-OH) pendant arms were investigated as members of a new family of efficient Ga(3+) chelators, TRAP ligands (triazacyclononane phosphinic acids). Stepwise protonation constants of ligands and stability constants of their complexes with Ga(3+), selected divalent metal, and Ln(3+) ions were determined by potentiometry. For comparison, equilibrium data for the metal ion-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) systems were redetermined. These ligands exhibit high thermodynamic selectivity for Ga(3+) over the other metal ions (log K(GaL) - log K(ML) = 7-9) and a selective complexation of smaller Mg(2+) over Ca(2+). Stabilities of the Ga(3+) complexes are dependent on the basicity of the donor atoms: [Ga(NOTA)] (log K(GaL) = 29.6) > [Ga(TRAP-OH)] (log K(GaL) = 23.3) > [Ga(TRAP-H)] (log K(GaL) = 21.9). The [Ga(TRAP-OH)] complex exhibits unusual reversible rearrangement of the "in-cage" N(3)O(3) complex to the "out-of-cage" O(6) complex. The in-cage complex is present in acidic solutions, and at neutral pH, Ga(3+) ion binds hydroxide anion, induces deprotonation and coordination of the P-hydroxymethyl group(s), and moves out of the macrocyclic cavity; the hypothesis is supported by a combination of results from potentiometry, multinuclear nuclear magnetic resonance spectrometry, and density functional theory calculations. Isomerism of the phosphinate Ga(3+) complexes caused by a combination of the chelate ring conformation, the helicity of coordinated pendant arms, and the chirality of the coordinated phosphinate groups was observed. All Ga(3+) complexes are kinetically inert in both acidic and alkaline solutions. Complex formation studies in acidic solutions indicate that Ga(3+) complexes of the phosphinate ligands are formed quickly (minutes) and quantitatively even at pH <2

  13. Kinetics of Social Contagion.

    PubMed

    Ruan, Zhongyuan; Iñiguez, Gerardo; Karsai, Márton; Kertész, János

    2015-11-20

    Diffusion of information, behavioral patterns or innovations follows diverse pathways depending on a number of conditions, including the structure of the underlying social network, the sensitivity to peer pressure and the influence of media. Here we study analytically and by simulations a general model that incorporates threshold mechanism capturing sensitivity to peer pressure, the effect of "immune" nodes who never adopt, and a perpetual flow of external information. While any constant, nonzero rate of dynamically introduced spontaneous adopters leads to global spreading, the kinetics by which the asymptotic state is approached shows rich behavior. In particular, we find that, as a function of the immune node density, there is a transition from fast to slow spreading governed by entirely different mechanisms. This transition happens below the percolation threshold of network fragmentation, and has its origin in the competition between cascading behavior induced by adopters and blocking due to immune nodes. This change is accompanied by a percolation transition of the induced clusters.

  14. Kinetics of Social Contagion

    NASA Astrophysics Data System (ADS)

    Ruan, Zhongyuan; Iñiguez, Gerardo; Karsai, Márton; Kertész, János

    2015-11-01

    Diffusion of information, behavioral patterns or innovations follows diverse pathways depending on a number of conditions, including the structure of the underlying social network, the sensitivity to peer pressure and the influence of media. Here we study analytically and by simulations a general model that incorporates threshold mechanism capturing sensitivity to peer pressure, the effect of "immune" nodes who never adopt, and a perpetual flow of external information. While any constant, nonzero rate of dynamically introduced spontaneous adopters leads to global spreading, the kinetics by which the asymptotic state is approached shows rich behavior. In particular, we find that, as a function of the immune node density, there is a transition from fast to slow spreading governed by entirely different mechanisms. This transition happens below the percolation threshold of network fragmentation, and has its origin in the competition between cascading behavior induced by adopters and blocking due to immune nodes. This change is accompanied by a percolation transition of the induced clusters.

  15. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  16. Ligand migration in sperm whale myoglobin.

    PubMed

    Scott, E E; Gibson, Q H

    1997-09-30

    Geminate oxygen rebinding to myoglobin was followed from a few nanoseconds to a few microseconds after photolysis for more than 25 different oxymyoglobin point mutants in the presence and absence of 12 atm of xenon. In all cases, two relaxations were observed: an initial fast phase (half-time 20 ns) and a slower, smaller phase (half-time 0.5-2 micros). Generally, xenon accelerates the fast reaction but slows the slower reaction and diminishes its amplitude. The rates and proportions of the two components and the effects of xenon on them vary widely for different mutants. The locations of specific xenon binding sites [Tilton, R. F., Kuntz, I. D. Jr., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857], the effects of point mutations on the geminate reactions, and molecular dynamics simulations were used to suggest locations in the protein interior occupied by ligands on the nanosecond to microsecond time scale. Photodissociated ligands may occupy xenon site 4 in the distal pocket and xenon site 1 below the plane of the heme. Rebinding from these positions corresponds to the slower geminate phase for O2 rebinding. The rapid geminate component is determined by competition between rebinding from a position closer to the iron atom and escape to solvent or more distant locations in the protein.

  17. Subnanometer Control of Mean Core Size during Mesofluidic Synthesis of Small (D(core) < 10 nm) Water-Soluble, Ligand-Stabilized Gold Nanoparticles.

    PubMed

    Elliott, Edward W; Haben, Patrick M; Hutchison, James E

    2015-11-03

    A convenient, single-step synthesis is reported that produces ligand-stabilized, water-soluble gold nanoparticles (AuNPs) with subnanometer-level precision of the mean core diameter over a range of 2-9 nm for a series of desired surface chemistries. The synthesis involves the reduction of a Au(III) species with sodium borohydride in the presence of a functionalized alkyl thiosulfate (Bunte salt) to yield thiolate-protected AuNPs. A key advantage of this synthesis is that simply adjusting the pH of the gold salt solution leads to control over the AuNP core size. The speciation of Au(III), and therefore the kinetics for its reduction and the core size produced, depends upon pH. The use of pH as the sole variable to control core size is a more reliable and convenient method than traditional approaches that rely on adjusting the concentrations and ratios of ligand, metal salt, and reducing agent. The average core size increased as the pH was raised for each ligand studied. Because the influence of pH was different for each of the ligands, working curves were plotted for each ligand to identify conditions to synthesize particles with specific, targeted core diameters. Using this approach, reaction conditions can be rapidly optimized using a combination of a mesofluidic reactor and small-angle X-ray scattering (SAXS) size analysis. The use of the mesofluidic reactor was needed to ensure fast mixing given the rapid kinetics for core formation. Using the reactor, it is possible to obtain reproducible sizes across multiple syntheses (<1-2% core size variation) and subnanometer control of the mean core dimensions. The synthetic method demonstrated here provides an attractive alternative to two-step syntheses involving ligand exchange because it is more efficient and eliminates the possibility of nanoparticle core size changes during exchange steps. This approach enables the development of "size ladders" of particles with the same surface chemistry for investigations of

  18. LASSO—ligand activity by surface similarity order: a new tool for ligand based virtual screening

    NASA Astrophysics Data System (ADS)

    Reid, Darryl; Sadjad, Bashir S.; Zsoldos, Zsolt; Simon, Aniko

    2008-06-01

    Virtual Ligand Screening (VLS) has become an integral part of the drug discovery process for many pharmaceutical companies. Ligand similarity searches provide a very powerful method of screening large databases of ligands to identify possible hits. If these hits belong to new chemotypes the method is deemed even more successful. eHiTS LASSO uses a new interacting surface point types (ISPT) molecular descriptor that is generated from the 3D structure of the ligand, but unlike most 3D descriptors it is conformation independent. Combined with a neural network machine learning technique, LASSO screens molecular databases at an ultra fast speed of 1 million structures in under 1 min on a standard PC. The results obtained from eHiTS LASSO trained on relatively small training sets of just 2, 4 or 8 actives are presented using the diverse directory of useful decoys (DUD) dataset. It is shown that over a wide range of receptor families, eHiTS LASSO is consistently able to enrich screened databases and provides scaffold hopping ability.

  19. Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary Constraints.

    PubMed

    Edman, Karl; Hosseini, Ali; Bjursell, Magnus K; Aagaard, Anna; Wissler, Lisa; Gunnarsson, Anders; Kaminski, Tim; Köhler, Christian; Bäckström, Stefan; Jensen, Tina J; Cavallin, Anders; Karlsson, Ulla; Nilsson, Ewa; Lecina, Daniel; Takahashi, Ryoji; Grebner, Christoph; Geschwindner, Stefan; Lepistö, Matti; Hogner, Anders C; Guallar, Victor

    2015-12-01

    Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.

  20. Tolrestat kinetics

    SciTech Connect

    Hicks, D.R.; Kraml, M.; Cayen, M.N.; Dubuc, J.; Ryder, S.; Dvornik, D.

    1984-10-01

    The kinetics of tolrestat, a potent inhibitor of aldose reductase, were examined. Serum concentrations of tolrestat and of total /sup 14/C were measured after dosing normal subjects and subjects with diabetes with /sup 14/C-labeled tolrestat. In normal subjects, tolrestat was rapidly absorbed and disappearance from serum was biphasic. Distribution and elimination t 1/2s were approximately 2 and 10 to 12 hr, respectively, after single and multiple doses. Unchanged tolrestat accounted for the major portion of /sup 14/C in serum. Radioactivity was rapidly and completely excreted in urine and feces in an approximate ratio of 2:1. Findings were much the same in subjects with diabetes. In normal subjects, the kinetics of oral tolrestat were independent of dose in the 10 to 800 mg range. Repetitive dosing did not result in unexpected cumulation. Tolrestat was more than 99% bound to serum protein; it did not compete with warfarin for binding sites but was displaced to some extent by high concentrations of tolbutamide or salicylate.

  1. Nanoscale Surface Curvature Effects on Ligand-Nanoparticle Interactions: A Plasmon-Enhanced Spectroscopic Study of Thiolated Ligand Adsorption, Desorption, and Exchange on Gold Nanoparticles.

    PubMed

    Villarreal, Esteban; Li, Guangfang Grace; Zhang, Qingfeng; Fu, Xiaoqi; Wang, Hui

    2017-07-12

    The interfacial adsorption, desorption, and exchange behaviors of thiolated ligands on nanotextured Au nanoparticle surfaces exhibit phenomenal site-to-site variations essentially dictated by the local surface curvatures, resulting in heterogeneous thermodynamic and kinetic profiles remarkably more sophisticated than those associated with the self-assembly of organothiol ligand monolayers on atomically flat Au surfaces. Here we use plasmon-enhanced Raman scattering as a spectroscopic tool combining time-resolving and molecular fingerprinting capabilities to quantitatively correlate the ligand dynamics with detailed molecular structures in real time under a diverse set of ligand adsorption, desorption, and exchange conditions at both equilibrium and nonequilibrium states, which enables us to delineate the effects of nanoscale surface curvature on the binding affinity, cooperativity, structural ordering, and the adsorption/desorption/exchange kinetics of organothiol ligands on colloidal Au nanoparticles. This work provides mechanistic insights on the key thermodynamic, kinetic, and geometric factors underpinning the surface curvature-dependent interfacial ligand behaviors, which serve as a central knowledge framework guiding the site-selective incorporation of desired surface functionalities into individual metallic nanoparticles for specific applications.

  2. INFLUENCE OF NATURAL AND SYNTHETIC ORGANIC LIGANDS ON THE STABILITY AND MOBILITY OF REDUCED TC(IV)

    SciTech Connect

    Nathalie A. Wall; Baohua Gu

    2012-12-20

    The primary objectives were (1) to quantify the interactions of organic ligands with Tc(IV) through the generation of thermodynamic (complexation) and kinetic parameters needed to assess and predict the mobility of reduced Tc(IV) at DOE contaminated sites; and (2) to determine the impact of organic ligands on the mobility and fate of reduced Tc(IV) under field geochemical conditions.

  3. Hybridization kinetics is different inside cells

    PubMed Central

    Schoen, Ingmar; Krammer, Hubert; Braun, Dieter

    2009-01-01

    It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solutions. Macromolecular crowding and specific binding interactions could change the diffusion properties and the availability of free molecules. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 μs to 1 s by a combination of laser-driven temperature oscillations and stroboscopic fluorescence imaging. The time constant of the hybridization depended on DNA concentration within individual cells and between cells. A quantitative analysis of the concentration dependence revealed several-fold accelerated kinetics as compared with free solution for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temperature oscillation optical lock-in microscopy allows to probe biomolecular interactions in different cell compartments in living cells for systems biology. PMID:20018715

  4. Bexarotene ligand pharmaceuticals.

    PubMed

    Hurst, R E

    2000-12-01

    Bexarotene (LGD-1069), from Ligand, was the first retinoid X receptor (RXR)-selective, antitumor retinoid to enter clinical trials. The company launched the drug for the treatment of cutaneous T-cell lymphoma (CTCL), as Targretin capsules, in the US in January 2000 [359023]. The company filed an NDA for Targretin capsules in June 1999, and for topical gel in December 1999 [329011], [349982] specifically for once-daily oral administration for the treatment of patients with early-stage CTCL who have not tolerated other therapies, patients with refractory or persistent early stage CTCL and patients with refractory advanced stage CTCL. The FDA approved Targretin capsules at the end of December 1999 for once-daily oral treatment of all stages of CTCL in patients refractory to at least one prior systemic therapy, at an initial dose of 300 mg/m2/day. After an NDA was submitted in December 1999 for Targretin gel, the drug received Priority Review status for use as a treatment of cutaneous lesions in patients with stage IA, IB or IIA CTCL [354836]. The FDA issued an approvable letter in June 2000, and granted marketing clearance for CTCL in the same month [370687], [372768], [372769], [373279]. Ligand had received Orphan Drug designation for this indication [329011]. At the request of the FDA, Ligand agreed to carry out certain post-approval phase IV and pharmacokinetic studies [351604]. The company filed an MAA with the EMEA for Targretin Capsules to treat lymphoma in November 1999 [348944]. The NDA for Targretin gel is based on a multicenter phase III trial that was conducted in the US, Canada, Europe and Australia involving 50 patients and a multicenter phase I/II clinical program involving 67 patients. Targretin gel was evaluated for the treatment of patients with early stage CTCL (IA-IIA) who were refractory to, intolerant to, or reached a response plateau for at least 6 months on at least two prior therapies. Efficacy results exceeded the protocol-defined response

  5. Formation of Foam-like Microstructural Carbon Material by Carbonization of Porous Coordination Polymers through a Ligand-Assisted Foaming Process.

    PubMed

    Kongpatpanich, Kanokwan; Horike, Satoshi; Fujiwara, Yu-Ichi; Ogiwara, Naoki; Nishihara, Hirotomo; Kitagawa, Susumu

    2015-09-14

    Porous carbon material with a foam-like microstructure has been synthesized by direct carbonization of porous coordination polymer (PCP). In situ generation of foaming agents by chemical reactions of ligands in PCP during carbonization provides a simple way to create lightweight carbon material with a foam-like microstructure. Among several substituents investigated, the nitro group has been shown to be the key to obtain the unique foam-like microstructure, which is due to the fast kinetics of gas evolution during carbonization. Foam-like microstructural carbon materials showed higher pore volume and specific capacitance compared to a microporous carbon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Kinetic buffers.

    PubMed

    Alibrandi, Giuseppe; Fabbrizzi, Luigi; Licchelli, Maurizio; Puglisi, Antonio

    2015-01-12

    This paper proposes a new type of molecular device that is able to act as an inverse proton sponge to slowly decrease the pH inside a reaction vessel. This makes the automatic monitoring of the concentration of pH-sensitive systems possible. The device is a composite formed of an alkyl chloride, which kinetically produces acidity, and a buffer that thermodynamically modulates the variation in pH value. Profiles of pH versus time (pH-t plots) have been generated under various experimental conditions by computer simulation, and the device has been tested by carrying out automatic spectrophotometric titrations, without using an autoburette. To underline the wide variety of possible applications, this new system has been used to realize and monitor HCl uptake by a di-copper(II) bistren complex in a single run, in a completely automatic experiment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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.

  8. Ligand binding analysis and screening by chemical denaturation shift.

    PubMed

    Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

    2013-12-01

    The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.

  9. Dynamics of catalytic resolution of 2-lithio-N-Boc-piperidine by ligand exchange.

    PubMed

    Beng, Timothy K; Tyree, William S; Parker, Trent; Su, Chicheung; Williard, Paul G; Gawley, Robert E

    2012-10-10

    The dynamics of the racemization and catalytic and stoichiometric dynamic resolution of 2-lithio-N-Boc-piperidine (7) have been investigated. The kinetic order in tetramethylethylenediamine (TMEDA) for both racemization and resolution of the title compound and the kinetic orders in two resolving ligands have been determined. The catalytic dynamic resolution is second order in TMEDA and 0.5 and 0.265 order in chiral ligands 8 and 10, respectively. The X-ray crystal structure of ligand 10 shows it to be an octamer. Dynamic NMR studies of the resolution process were carried out. Some of the requirements for a successful catalytic dynamic resolution by ligand exchange have been identified.

  10. EDTA modified LDHs as Cu2+ scavengers: removal kinetics and sorbent stability.

    PubMed

    Rojas, Ricardo; Perez, M Rosario; Erro, Eustaquio M; Ortiz, Patricia I; Ulibarri, Maria Angeles; Giacomelli, Carla E

    2009-03-15

    EDTA modified layered double hydroxides (LDHs) were investigated as potential sorbents to remediate heavy metals pollution. The polidentate ligand was introduced by an exchange method in a Zn-Al-LDH, which takes place with partial erosion of the layers, causing the intercalation of [Zn(EDTA)](2-) complex instead of the ligand. [Cu(H(2)O)(6)](2+) cation was selected as a model cation to study the uptake mechanism, exploring the elimination kinetics from the first minutes up to the steady state. A flow injection analysis system coupled to an amperometric detector (FIA-AM) was applied to perform fast and reliable [Cu(H(2)O)(6)](2+) determinations in monodisperse solid-aqueous solution systems. Furthermore, the sorbent stability was determined as a function of the pH and the nitrate concentration. The [Cu(H(2)O)(6)](2+) elimination is produced by an exchange reaction with [Zn(EDTA)](2-) anions placed either in the solid interlayer or in the aqueous solution, this last being released from the sorbent. Additional [Cu(H(2)O)(6)](2+) removal is produced by Cu(OH)(2) precipitation at high copper concentrations due to the LDHs high pH buffering capacity. The sorbent removes [Cu(H(2)O)(6)](2+) with high affinity in a wide concentration range. The elimination process reaches equilibrium in less than 30 min and leaves metal cation concentrations lower than 0.05 ppm in the supernatants.

  11. Measurement of Ligand-Target Residence Times by (1)H Relaxation Dispersion NMR Spectroscopy.

    PubMed

    Moschen, Thomas; Grutsch, Sarina; Juen, Michael A; Wunderlich, Christoph H; Kreutz, Christoph; Tollinger, Martin

    2016-12-08

    A ligand-observed (1)H NMR relaxation experiment is introduced for measuring the binding kinetics of low-molecular-weight compounds to their biomolecular targets. We show that this approach, which does not require any isotope labeling, is applicable to ligand-target systems involving proteins and nucleic acids of variable molecular size. The experiment is particularly useful for the systematic investigation of low affinity molecules with residence times in the micro- to millisecond time regime.

  12. Ligand binding and proton exchange dynamics in site-specific mutants of human myoglobin

    SciTech Connect

    Lambright, D.G.

    1992-01-01

    Site specific mutagenesis was used to make substitutions of four residues in the distal heme pocket of human myoglobin: Val68, His64, Lys45, and Asp60. Strongly diffracting crystals of the conservative mutation K45R in the met aquo form were grown in the trigonal space group P3[sub 2]21 and the X-ray crystal structure determined at 1.6 [angstrom] resolution. The overall structure is similar to that of sperm whale met aquo myoglobin. Several of the mutant proteins were characterized by 2-D NMR spectroscopy. The NMR data suggest the structural changes are localized to the region of the mutation. The dynamics of ligand binding to myoglobin mutants were studied by transient absorption spectroscopy following photolysis of the CO complexes. Transient absorption kinetics and spectra on the ns to ms timescale were measured in aqueous solution from 280 K to 310 K and in 75% glycerol: water from 250 K to 310 K. Two significant basis spectra were obtained from singular value decomposition of the matrix of time dependent spectra. The information was used to obtain approximations for the extent of ligand rebinding and the kinetics of conformational relaxation. Except for K45R, substitutions at Lys45 or Asp60 produce changes in the kinetics for ligand rebinding. Replacement of Lys45 with Arg increases the rate of ligand rebinding from the protein matrix by a factor of 2, but does not alter the rates for ligand escape or entry into the protein or the dynamics of the conformational relaxation. Substitutions at His64 and Val68 influence the kinetics of ligand rebinding and the dynamics of conformational relaxation. The results do not support the hypothesis that ligand migration between the heme pocket and solvent is determined solely by fluctuations of Arg45 and His64 between open and closed conformations of the heme pocket but can be rationalized if ligand diffusion through the protein matrix involves multiple competing pathways.

  13. A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.

    PubMed

    Isom, Daniel G; Marguet, Philippe R; Oas, Terrence G; Hellinga, Homme W

    2011-04-01

    Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.

  14. FAST: FAST Analysis of Sequences Toolbox

    PubMed Central

    Lawrence, Travis J.; Kauffman, Kyle T.; Amrine, Katherine C. H.; Carper, Dana L.; Lee, Raymond S.; Becich, Peter J.; Canales, Claudia J.; Ardell, David H.

    2015-01-01

    FAST (FAST Analysis of Sequences Toolbox) provides simple, powerful open source command-line tools to filter, transform, annotate and analyze biological sequence data. Modeled after the GNU (GNU's Not Unix) Textutils such as grep, cut, and tr, FAST tools such as fasgrep, fascut, and fastr make it easy to rapidly prototype expressive bioinformatic workflows in a compact and generic command vocabulary. Compact combinatorial encoding of data workflows with FAST commands can simplify the documentation and reproducibility of bioinformatic protocols, supporting better transparency in biological data science. Interface self-consistency and conformity with conventions of GNU, Matlab, Perl, BioPerl, R, and GenBank help make FAST easy and rewarding to learn. FAST automates numerical, taxonomic, and text-based sorting, selection and transformation of sequence records and alignment sites based on content, index ranges, descriptive tags, annotated features, and in-line calculated analytics, including composition and codon usage. Automated content- and feature-based extraction of sites and support for molecular population genetic statistics make FAST useful for molecular evolutionary analysis. FAST is portable, easy to install and secure thanks to the relative maturity of its Perl and BioPerl foundations, with stable releases posted to CPAN. Development as well as a publicly accessible Cookbook and Wiki are available on the FAST GitHub repository at https://github.com/tlawrence3/FAST. The default data exchange format in FAST is Multi-FastA (specifically, a restriction of BioPerl FastA format). Sanger and Illumina 1.8+ FastQ formatted files are also supported. FAST makes it easier for non-programmer biologists to interactively investigate and control biological data at the speed of thought. PMID:26042145

  15. Melatonin: functions and ligands.

    PubMed

    Singh, Mahaveer; Jadhav, Hemant R

    2014-09-01

    Melatonin is a chronobiotic substance that acts as synchronizer by stabilizing bodily rhythms. Its synthesis occurs in various locations throughout the body, including the pineal gland, skin, lymphocytes and gastrointestinal tract (GIT). Its synthesis and secretion is controlled by light and dark conditions, whereby light decreases and darkness increases its production. Thus, melatonin is also known as the 'hormone of darkness'. Melatonin and analogs that bind to the melatonin receptors are important because of their role in the management of depression, insomnia, epilepsy, Alzheimer's disease (AD), diabetes, obesity, alopecia, migraine, cancer, and immune and cardiac disorders. In this review, we discuss the mechanism of action of melatonin in these disorders, which could aid in the design of novel melatonin receptor ligands.

  16. Formation of very stable and selective Cu(II) complexes with a non-macrocyclic ligand: can basicity rival pre-organization?

    PubMed

    Abada, Sabah; Lecointre, Alexandre; Elhabiri, Mourad; Charbonnière, Loïc J

    2010-10-14

    The synthesis of ligand L based on a 2,6-bis[(N,N-bis(methylene phosphonic acid)aminomethyl] pyridine scaffold is described. Potentiometry combined with UV-Vis absorption spectrophotometric titrations were used to determine the protonation constants of the ligand and the stability constants of its corresponding Cu(II), Ni(II), Zn(II) and Ga(III) cations (0.1 M NaClO(4), 25.0 °C). The physico-chemical approach revealed very large stability constants for Cu(II) complexation (logK(CuL) = 22.71(7)) reflected in a very high pCu(II) value of ∼ 15.5 (pH = 7.4, [L](tot) = 10(-5) M, [Cu](tot) = 10(-6) M), close to those measured for the strong methylphosphonate functionalized cyclen chelators. Based on a literature survey, a correlation is proposed between the pK values of branched polyamine ligands and their stability constants for Cu(II) complexation, allowing for an estimation of the latter on the basis of the protonation constants of L. Ligand L was also shown to be very selective towards Cu(II) compared to the other cations studied (ΔlogK > 4). UV-Vis spectroscopy and kinetic measurements indicated that the formation of the cupric complexes with L is very fast, which, in combination with all other properties, makes it an excellent non-cyclic target for Cu(II) radiopharmaceutical within the frame of (64)Cu positron emission tomography imaging and radiotherapy.

  17. Ligand binding to a high-energy partially unfolded protein.

    PubMed

    Kasper, Joseph R; Park, Chiwook

    2015-01-01

    The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP(+) as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP(+) and found that NADP(+) binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP(+) is diminished upon partial unfolding. Based on known crystallographic structures of NADP(+) -bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine-binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP(+) . Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high-energy non-native forms.

  18. The Chemistry of Separations Ligand Degradation by Organic Radical Cations

    SciTech Connect

    Mezyk, Stephen P.; Horne, Gregory P.; Mincher, Bruce J.; Zalupski, Peter R.; Cook, Andrew R.; Wishart, James F.

    2016-12-01

    Solvent based extractions of used nuclear fuel use designer ligands in an organic phase extracting ligand complexed metal ions from an acidic aqueous phase. These extractions will be performed in highly radioactive environments, and the radiation chemistry of all these complexants and their diluents will play a major role in determining extraction efficiency, separation factors, and solvent-recycle longevity. Although there has been considerable effort in investigating ligand damage occurring in acidic water radiolysis conditions, only minimal fundamental kinetic and mechanistic data has been reported for the degradation of extraction ligands in the organic phase. Extraction solvent phases typically use normal alkanes such as dodecane, TPH, and kerosene as diluents. The radiolysis of such diluents produce a mixture of radical cations (R•+), carbon-centered radicals (R), solvated electrons, and molecular products such as hydrogen. Typically, the radical species will preferentially react with the dissolved oxygen present to produce relatively inert peroxyl radicals. This isolates the alkane radical cation species, R•+ as the major radiolytically-induced organic species that can react with, and degrade, extraction agents in this phase. Here we report on our recent studies of organic radical cation reactions with various ligands. Elucidating these parameters, and combining them with the known acidic aqueous phase chemistry, will allow a full, fundamental, understanding of the impact of radiation on solvent extraction based separation processes to be achieved.

  19. Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods.

    PubMed

    Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

    2016-01-26

    Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein-ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein-ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models--the "lock-and-key", "induced fit", and "conformational selection"--are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein-ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed.

  20. The Chemistry of Separations Ligand Degradation by Organic Radical Cations

    DOE PAGES

    Mezyk, Stephen P.; Horne, Gregory P.; Mincher, Bruce J.; ...

    2016-12-01

    Solvent based extractions of used nuclear fuel use designer ligands in an organic phase extracting ligand complexed metal ions from an acidic aqueous phase. These extractions will be performed in highly radioactive environments, and the radiation chemistry of all these complexants and their diluents will play a major role in determining extraction efficiency, separation factors, and solvent-recycle longevity. Although there has been considerable effort in investigating ligand damage occurring in acidic water radiolysis conditions, only minimal fundamental kinetic and mechanistic data has been reported for the degradation of extraction ligands in the organic phase. Extraction solvent phases typically use normalmore » alkanes such as dodecane, TPH, and kerosene as diluents. The radiolysis of such diluents produce a mixture of radical cations (R•+), carbon-centered radicals (R•), solvated electrons, and molecular products such as hydrogen. Typically, the radical species will preferentially react with the dissolved oxygen present to produce relatively inert peroxyl radicals. This isolates the alkane radical cation species, R•+ as the major radiolytically-induced organic species that can react with, and degrade, extraction agents in this phase. Here we report on our recent studies of organic radical cation reactions with various ligands. Elucidating these parameters, and combining them with the known acidic aqueous phase chemistry, will allow a full, fundamental, understanding of the impact of radiation on solvent extraction based separation processes to be achieved.« less

  1. Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods

    PubMed Central

    Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

    2016-01-01

    Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed. PMID:26821017

  2. Protein ligand interactions. Part 5: Isoquinoline alkaloids as inhibitors of acetylcholinesterase from Electrophorus electricus.

    PubMed

    Whiteley, C G; Daya, S

    1995-01-01

    Kinetic analysis has shown that papaverine, berberine and isoquinoline alkaloids acts as reversible competitive inhibitors of acetylcholinesterase with respect to the substrate, acetylthiocholine chloride. The inhibitor constants (Ki) vary from 3.5 microM to 88 microM. With time they act as irreversible covalent inhibitors with papaverine producing 85% inactivation after 40 min. Pseudo first-order kinetics are observed with the rate constant being proportional to the concentration of the ligand and the order of reaction being equal to one. Spectrophotometry was used to study the binding of the ligands with acetylcholinesterase and Scatchard analysis used to calculate the respective dissociation constants and the number of binding sites.

  3. Fluorescence‐ and bioluminescence‐based approaches to study GPCR ligand binding

    PubMed Central

    Stoddart, Leigh A; White, Carl W; Nguyen, Kim; Hill, Stephen J

    2015-01-01

    Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd) and competition (Ki) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor‐associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time‐resolved FRET (TR‐FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super‐bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein‐Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc PMID:26317175

  4. Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.

    PubMed

    Stoddart, Leigh A; White, Carl W; Nguyen, Kim; Hill, Stephen J; Pfleger, Kevin D G

    2016-10-01

    Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd ) and competition (Ki ) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super-bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc. © 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on

  5. Photolytic degradation of methylmercury enhanced by binding to natural organic ligands

    PubMed Central

    Zhang, Tong; Hsu-Kim, Heileen

    2010-01-01

    Monomethylmercury is a neurotoxin that poses significant risks to human health1 due to its bioaccumulation in food webs. Sunlight degradation to inorganic mercury is an important component of the mercury cycle that maintains methylmercury at low concentrations in natural waters. Rates of photodecomposition, however, can vary drastically between surface waters2–5 for reasons that are largely unknown. Here, we show that photodegradation occurs through singlet oxygen, a highly reactive form of dissolved oxygen generated by sunlight irradiation of dissolved natural organic matter. The kinetics of degradation, however, depended on water constituents that bind methylmercury cations. Relatively fast degradation rates (similar to observations in freshwater lakes) applied only to methylmercury species bound to organic sulfur-containing thiol ligands such as glutathione, mercaptoacetate, and humics. In contrast, methylmercury-chloride complexes, which are dominant in marine systems, were unreactive. Binding by thiols lowered the excitation energy of the carbon-mercury bond on the methylmercury molecule6–7 and subsequently increased reactivity towards bond breakage and decomposition. Our results explain methylmercury photodecomposition rates that are relatively rapid in freshwater lakes2–4 and slow in marine waters5. PMID:20634995

  6. Navigating ligand protein binding free energy landscapes: universality and diversity of protein folding and molecular recognition mechanisms

    NASA Astrophysics Data System (ADS)

    Verkhivker, Gennady M.; Rejto, Paul A.; Bouzida, Djamal; Arthurs, Sandra; Colson, Anthony B.; Freer, Stephan T.; Gehlhaar, Daniel K.; Larson, Veda; Luty, Brock A.; Marrone, Tami; Rose, Peter W.

    2001-03-01

    Thermodynamic and kinetic aspects of ligand-protein binding are studied for the methotrexate-dihydrofolate reductase system from the binding free energy profile constructed as a function of the order parameter. Thermodynamic stability of the native complex and a cooperative transition to the unique native structure suggest the nucleation kinetic mechanism at the equilibrium transition temperature. Structural properties of the transition state ensemble and the ensemble of nucleation conformations are determined by kinetic simulations of the transmission coefficient and ligand-protein association pathways. Structural analysis of the transition states and the nucleation conformations reconciles different views on the nucleation mechanism in protein folding.

  7. Bifunctional DTPA-type ligand

    SciTech Connect

    Gansow, O.A.; Brechbiel, M.W.

    1990-03-26

    The subject matter of the invention relates to bifunctional cyclohexyl DTPA ligands and methods of using these compounds. Specifically, such ligands are useful for radiolabeling proteins with radioactive metals, and can consequently be utilized with respect to radioimmunoimaging and/or radioimmunotherapy.

  8. Al(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Sodupe, M.; Bauschlicher, Charles W., Jr.

    1991-01-01

    Ab initio calculations are used to optimize the structure and determine the binding energies of Al(+) to a series of ligands. For Al(+)-CN, the bonding was found to have a large covalent component. For the remaining ligands, the bonding is shown to be electrostatic in origin. The results obtained for Al(+) are compared with those previously reported for Mg(+).

  9. Cloud computing approaches for prediction of ligand binding poses and pathways

    PubMed Central

    Lawrenz, Morgan; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    We describe an innovative protocol for ab initio prediction of ligand crystallographic binding poses and highly effective analysis of large datasets generated for protein-ligand dynamics. We include a procedure for setup and performance of distributed molecular dynamics simulations on cloud computing architectures, a model for efficient analysis of simulation data, and a metric for evaluation of model convergence. We give accurate binding pose predictions for five ligands ranging in affinity from 7 nM to > 200 μM for the immunophilin protein FKBP12, for expedited results in cases where experimental structures are difficult to produce. Our approach goes beyond single, low energy ligand poses to give quantitative kinetic information that can inform protein engineering and ligand design. PMID:25608737

  10. Ligand dissociation mediated charge transfer observed at colloidal W18O49 nanoparticle interfaces.

    PubMed

    Grauer, David C; Alivisatos, A Paul

    2014-03-11

    Understanding charge transfer dynamics through the ligand shell of colloidal nanoparticles has been an important pursuit in solar energy conversion. While charge transport through ligand shells of nanoparticle films has been studied intensely in static dry and electrochemical systems, its influence on charge transfer kinetics in dispersed colloidal systems has received relatively less attention. This work reports the oxidation of amine passivated tungsten oxide nanoparticles by an organically soluble tris-(1,10-phenanthroline) iron(III) derivative. By following the rate of this oxidation optically via the production of the ferroin derivative under various reaction conditions and particle derivatizations, we are able to show that the fluxional ligand shells on dispersed, colloidal nanoparticles provide a separate and more facile pathway for charge transfer, in which the rate-limiting step for charge transfer is the ligand dissociation. Since such ligand shells are frequently required for nanoparticle stability, this observation has significant implications for colloidal nanoparticle photocatalysis.

  11. Distribution kinetics of dietary methylmercury in the arctic charr (Salvelinus alpinus)

    SciTech Connect

    Ribeiro, C.A.O.; Rouleau, C.; Pelletier, E.; Audet, C.; Tjaelve, H.

    1999-03-15

    The authors fed immature 1+ arctic charr with a single dose of methyl[{sup 203}Hg]mercury (MeHg) and quantified distribution kinetics with a new and simple three-compartment caternary model having well-perfused viscera and blood as the central compartment (VB), whereas gut (G) and the rest of body (R) constituted the peripheral compartments. The model accurately described distribution kinetics of MeHg in the fish, using either data of MeHg content in compartments or blood concentration data. Despite the known fast translocation of MeHg between binding sites at the molecular level, its translocation rate between compartments was surprisingly slow, 27 days being needed to complete 95% of the transfer from gut to blood and 48 days for the subsequent transfer to compartment R. This probably results from a limitation of the stepwise transfer rate of MeHg from red blood cells, which contain most of blood MeHg, to plasma and then to tissues due to low plasmatic concentration of small mobile sulfhydryl ligands. The model presented is a convenient tool that could be used to compare the fate of MeHg and other organometals, such as butyltins and alkylleads, in various aquatic and terrestrial animal species.

  12. Atomic level computational identification of ligand migration pathways between solvent and binding site in myoglobin.

    PubMed

    Ruscio, Jory Z; Kumar, Deept; Shukla, Maulik; Prisant, Michael G; Murali, T M; Onufriev, Alexey V

    2008-07-08

    Myoglobin is a globular protein involved in oxygen storage and transport. No consensus yet exists on the atomic level mechanism by which oxygen and other small nonpolar ligands move between the myoglobin's buried heme, which is the ligand binding site, and surrounding solvent. This study uses room temperature molecular dynamics simulations to provide a complete atomic level picture of ligand migration in myoglobin. Multiple trajectories--providing a cumulative total of 7 micros of simulation--are analyzed. Our simulation results are consistent with and tie together previous experimental findings. Specifically, we characterize: (i) Explicit full trajectories in which the CO ligand shuttles between the internal binding site and the solvent and (ii) pattern and structural origins of transient voids available for ligand migration. The computations are performed both in sperm whale myoglobin wild-type and in sperm whale V68F myoglobin mutant, which is experimentally known to slow ligand-binding kinetics. On the basis of these independent, but mutually consistent ligand migration and transient void computations, we find that there are two discrete dynamical pathways for ligand migration in myoglobin. Trajectory hops between these pathways are limited to two bottleneck regions. Ligand enters and exits the protein matrix in common identifiable portals on the protein surface. The pathways are located in the "softer" regions of the protein matrix and go between its helices and in its loop regions. Localized structural fluctuations are the primary physical origin of the simulated CO migration pathways inside the protein.

  13. 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.

  14. Ligand Lone-Pair Influence on Hydrocarbon C-H Activation. A Computational Perspective

    SciTech Connect

    Ess, Daniel H.; Gunnoe, T. Brent; Cundari, Thomas R.; Goddard, William A.; Periana, Roy A.

    2010-12-03

    Mid to late transition metal complexes that break hydrocarbon C-H bonds by transferring the hydrogen to a heteroatom ligand while forming a metal-alkyl bond offer a promising strategy for C-H activation. Here we report a density functional (B3LYP, M06, and X3LYP) analysis of cis-(acac)2MX and TpM(L)X (M = Ir, Ru, Os, and Rh; acac = acetylacetonate, Tp = tris(pyrazolyl)borate; X = CH3, OH, OMe, NH2, and NMe2) systems for methane C-H bond activation reaction kinetics and thermodynamics. We address the importance of whether a ligand lone pair provides an intrinsic kinetic advantage through possible electronic dπ-pπ repulsions for M-OR and M-NR2 systems versus M-CH3 systems. This involves understanding the energetic impact of the X ligand group on ligand loss, C-H bond coordination, and C-H bond cleavage steps as well as understanding how the nucleophilicity of the ligand X group, the electrophilicity of the transition metal center, and cis-ligand stabilization effect influence each of these steps. We also explore how spectator ligands and second- versus third-row transition metal centers impact the energetics of each of these C-H activation steps.

  15. Stability of Phosphine-Ligated Gold Cluster Ions toward Dissociation: Effect of Ligand and Cluster Size

    NASA Astrophysics Data System (ADS)

    Laskin, Julia

    2015-03-01

    Precise control of the composition of phosphine-ligated gold clusters is of interest to their applications in catalysis, sensing, and drug delivery. Reduction synthesis in solution typically generates a distribution of ligated clusters containing different number of gold atoms and capping ligands. Ligand binding energy is an important factor determining the kinetics of cluster nucleation and growth in solution and hence the resulting cluster distribution. Phosphines are popular capping ligands with tunable electronic and steric properties that affect their binding to the gold core. We examined the effect of the number of gold atoms in the cluster and the properties of the phosphine ligand on the ligand binding energy to the gold core using surface-induced dissociation (SID) of mass selected cluster cations produced through electrospray ionization. SID of vibrationally excited ions is ideally suited for studying gas-phase fragmentation of complex ions such as ligated gold clusters. The energetics, dynamics, and mechanisms of cluster ion fragmentation in the absence of solvent are determined through RRKM modeling of time and kinetic energy dependent SID spectra. This approach provides quantitative information on the ligand binding energies in phosphine-ligated gold clusters important for understanding their formation in solution. Furthermore, ligand binding energies derived from SID data provide the first benchmark values for comparison with electronic structure calculations. This work was supported by the US Department of Energy (DOE), Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences.

  16. Simulation of the kinetics of oxygen complexes in crystalline silicon

    NASA Astrophysics Data System (ADS)

    Joo Lee, Young; von Boehm, J.; Nieminen, R. M.

    2002-10-01

    The formation kinetics of thermal double donors (TDD's) is studied by a general kinetic model with parameters based on accurate ab initio total-energy calculations. The kinetic model includes all relevant association, dissociation, and restructuring processes. The simulated kinetics agrees qualitatively and in most cases quantitatively with the experimentally found consecutive kinetics of TDD's. It also supports our earlier assignments of the ring-type oxygen chains to TDD's [Pesola et al., Phys. Rev. Lett. 84, 5343 (2000)]. We demonstrate with the kinetic model that the most common assumption that only the O2 dimer acts as a fast diffusing species would lead to an unrealistic steady increase of the concentration of O3. The neglect of restructuring processes leads to an anomalous increase of oxygen dimers and negligible concentrations of TDD's. The capture of interstitial oxygens by diffusing oxygen chains and the escaping of interstitial oxygens from the chains fully dominate the formation kinetics.

  17. Protein-Ligand Docking Based on Beta-Shape

    NASA Astrophysics Data System (ADS)

    Kim, Chong-Min; Won, Chung-In; Kim, Jae-Kwan; Ryu, Joonghyun; Bhak, Jong; Kim, Deok-Soo

    Protein-ligand docking is to predict the location and orientation of a ligand with respect to a protein within its binding site, and has been known to be essential for the development of new drugs. The protein-ligand docking problem is usually formulated as an energy minimization problem to identify the docked conformation of the ligand. A ligand usually docks around a depressed region, called a pocket, on the surface of a protein. Presented in this paper is a docking method, called BetaDock, based on the newly developed geometric construct called the β-shape and the β-complex. To cope with the computational intractability, the global minimum of the potential energy function is searched using the genetic algorithm. The proposed algorithm first locates initial chromosomes at some locations within the pocket recognized according to the local shape of the β-shape. Then, the algorithm proceeds generations by taking advantage of powerful properties of the β-shape to achieve an extremely fast and good solution. We claim that the proposed method is much faster than other popular docking softwares including AutoDock.

  18. Conformational kinetics reveals affinities of protein conformational states.

    PubMed

    Daniels, Kyle G; Suo, Yang; Oas, Terrence G

    2015-07-28

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

  19. Conformational kinetics reveals affinities of protein conformational states

    PubMed Central

    Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.

    2015-01-01

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states. PMID:26162682

  20. How hydrophobic drying forces impact the kinetics of molecular recognition.

    PubMed

    Mondal, Jagannath; Morrone, Joseph A; Berne, B J

    2013-08-13

    A model of protein-ligand binding kinetics, in which slow solvent dynamics results from hydrophobic drying transitions, is investigated. Molecular dynamics simulations show that solvent in the receptor pocket can fluctuate between wet and dry states with lifetimes in each state that are long enough for the extraction of a separable potential of mean force and wet-to-dry transitions. We present a diffusive surface hopping model that is represented by a 2D Markovian master equation. One dimension is the standard reaction coordinate, the ligand-pocket separation, and the other is the solvent state in the region between ligand and binding pocket which specifies whether it is wet or dry. In our model, the ligand diffuses on a dynamic free-energy surface which undergoes kinetic transitions between the wet and dry states. The model yields good agreement with results from explicit solvent molecular dynamics simulation and an improved description of the kinetics of hydrophobic assembly. Furthermore, it is consistent with a "non-Markovian Brownian theory" for the ligand-pocket separation coordinate alone.

  1. Triple bioaffinity mass spectrometry concept for thyroid transporter ligands.

    PubMed

    Aqai, Payam; Fryganas, Christos; Mizuguchi, Mineyuki; Haasnoot, Willem; Nielen, Michel W F

    2012-08-07

    For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone (13)C(6)-L-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model "unknown" EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.

  2. Ligand Docking and Virtual Screening in Structure-based Drug Discovery

    NASA Astrophysics Data System (ADS)

    Cavasotto, Claudio N.

    2006-08-01

    As the number of high-resolution three-dimensional protein and nucleic acid structures continues to grow, ligand-docking—based virtual screening of chemical libraries to a receptor are playing a critical role in the drug discovery process by identifying new `drug-candidates'. The capability to correctly predict ligand-protein interaction is fundamental to any accurate docking algorithm and the necessary starting point for any reliable virtual screening protocol. Furthermore, explicit consideration of receptor flexibility in computational ligand docking is emerging in many cases as crucial for an accurate prediction of the orientation and interactions of ligands within the binding pocket. The combination of ligand docking with a fast scoring algorithm that can account for the thermodynamics of binding, and discriminate between potential active/inactive compounds, can greatly reduced the number of compounds to be tested experimentally, while predicting a detailed structure of hits bound to the receptor useful enough to help the synthetic elaboration of leads.

  3. Coupled hydro-neutronic calculations for fast burst reactor accidents

    SciTech Connect

    Paternoster, R.; Kimpland, R.; Jaegers, P.; McGhee, J.

    1994-01-01

    Methods are described for determining the fully coupled neutronic/hydrodynamic response of fast burst reactors (FBR) under disruptive accident conditions. Two code systems, PAD (1 -D Lagrangian) and NIKE-PAGOSA (3-D Eulerian) were used to accomplish this. This is in contrast to the typical methodology that computes these responses by either single point kinetics or in a decoupled manner. This methodology is enabled by the use of modem supercomputers (CM-200). Two examples of this capability are presented: an unreflected metal fast burst assembly, and a reflected fast burst assembly typical of the Skua or SPR-III class of fast burst reactor.

  4. Kinetic behavior of lanthanide extraction with acidic phosphorus extractants

    SciTech Connect

    Ma, E.; Muralidharan, S.; Freiser, H.

    1995-12-01

    The kinetics of the formation of the complexes of tervalent lanthanides with several organophosphorus extractants in the octane-H{sub 2}O phase pair have been characterized by competition kinetics. The kinetic studies were conducted by the extraction of the EDTA complexes of Y{sup 3+}, Nd{sup 3+}, Gd{sup 3+}, Ho{sup 3+} and Lu{sup 3+} by bis(2-ethylhexyl)phosphoric acid (HDEHP), 2-ethylhexylphosphonic acid mono-2-ethylhexyl ester (HEH[EHP]) and bis(2,4,4-trimethylpentyl)phosphinic acid (Cyanex 272) in octane. The observed rate constants bore an inverse relationship to the stability constants of the metal-EDTA complexes. For a given metal the rate constants as a function of ligand followed the order HDEHP > HEH[EHP] > Cyanex 272. The mechanistic details of the competition kinetics and the implications of the kinetic results will be discussed.

  5. Protein ligand interaction database (PLID).

    PubMed

    Reddy, A Srinivas; Amarnath, H S Durga; Bapi, Raju S; Sastry, G Madhavi; Sastry, G Narahari

    2008-10-01

    A comprehensive database named, protein ligand interaction database (PLID), is created with 6295 ligands bound to proteins extracted from the protein data bank (PDB). This is by far the most comprehensive database of physico-chemical properties, quantum mechanical descriptors and the residues present in the active site of proteins. It is a publicly available web-based database (via the Internet) at http://203.199.182.73/gnsmmg/databases/plid/.

  6. Validity of ligand efficiency metrics.

    PubMed

    Murray, Christopher W; Erlanson, Daniel A; Hopkins, Andrew L; Keserü, György M; Leeson, Paul D; Rees, David C; Reynolds, Charles H; Richmond, Nicola J

    2014-06-12

    A recent viewpoint article (Improving the plausibility of success with inefficient metrics. ACS Med. Chem. Lett. 2014, 5, 2-5) argued that the standard definition of ligand efficiency (LE) is mathematically invalid. In this viewpoint, we address this criticism and show categorically that the definition of LE is mathematically valid. LE and other metrics such as lipophilic ligand efficiency (LLE) can be useful during the multiparameter optimization challenge faced by medicinal chemists.

  7. Mechanism of activation of a hafnium pyridyl-amide olefin polymerization catalyst: ligand modification by monomer.

    PubMed

    Froese, Robert D J; Hustad, Phillip D; Kuhlman, Roger L; Wenzel, Timothy T

    2007-06-27

    . Experimental evidence for aryl-insertion products is obtained from reactions of ethylene (13C2H4 NMR studies) or 4-methyl-1-pentene (4M1P) using relatively low monomer/catalyst ratios. Quantitative generation of monomer-inserted products is complicated by slow initiation kinetics followed by fast polymerization kinetics. However, NMR evidence for reaction with 13C2H4 was observed in situ at low temperature, and the attachment of monomer to ligand was confirmed by GC/MS and 13C NMR after quenching. Furthermore, a 4M1P-appended ligand was isolated from a polymerization reaction (50:1 monomer:catalyst) by column chromatography followed by multiple recrystallizations. One isomer was characterized by X-ray crystallography, which unequivocally shows a 4-methylpentyl substituent at the 2-position of the naphthyl, consistent with 1,2-insertion into the Hf-aryl bond. NMR suggests a second diastereomer (not isolated) is formed from a 1,2-insertion of opposite stereoselectivity.

  8. Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

    PubMed

    Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

    2016-05-09

    Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

  9. Chemical and Biological Kinetics

    NASA Astrophysics Data System (ADS)

    Emanuel', N. M.

    1981-10-01

    Examples of the application of the methods and ideas of chemical kinetics in various branches of chemistry and biology are considered and the results of studies on the kinetics and mechanisms of autoxidation and inhibited and catalysed oxidation of organic substances in the liquid phase are surveyed. Problems of the kinetics of the ageing of polymers and the principles of their stabilisation are discussed and certain trends in biological kinetics (kinetics of tumour growth, kinetic criteria of the effectiveness of chemotherapy, problems of gerontology, etc.) are considered. The bibliography includes 281 references.

  10. Ligand Identification Scoring Algorithm (LISA)

    PubMed Central

    Zheng, Zheng; Merz, Kenneth M.

    2011-01-01

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms. PMID:21561101

  11. Ligand Identification Scoring Algorithm (LISA).

    PubMed

    Zheng, Zheng; Merz, Kenneth M

    2011-06-27

    A central problem in de novo drug design is determining the binding affinity of a ligand with a receptor. A new scoring algorithm is presented that estimates the binding affinity of a protein-ligand complex given a three-dimensional structure. The method, LISA (Ligand Identification Scoring Algorithm), uses an empirical scoring function to describe the binding free energy. Interaction terms have been designed to account for van der Waals (VDW) contacts, hydrogen bonding, desolvation effects, and metal chelation to model the dissociation equilibrium constants using a linear model. Atom types have been introduced to differentiate the parameters for VDW, H-bonding interactions, and metal chelation between different atom pairs. A training set of 492 protein-ligand complexes was selected for the fitting process. Different test sets have been examined to evaluate its ability to predict experimentally measured binding affinities. By comparing with other well-known scoring functions, the results show that LISA has advantages over many existing scoring functions in simulating protein-ligand binding affinity, especially metalloprotein-ligand binding affinity. Artificial Neural Network (ANN) was also used in order to demonstrate that the energy terms in LISA are well designed and do not require extra cross terms.

  12. Synthesis and Base Hydrolysis of a Cobalt(III) Complex Coordinated by a Thioether Ligand

    ERIC Educational Resources Information Center

    Roecker, Lee

    2008-01-01

    A two-week laboratory experiment for students in advanced inorganic chemistry is described. Students prepare and characterize a cobalt(III) complex coordinated by a thioether ligand during the first week of the experiment and then study the kinetics of Co-S bond cleavage in basic solution during the second week. The synthetic portion of the…

  13. Synthesis and Base Hydrolysis of a Cobalt(III) Complex Coordinated by a Thioether Ligand

    ERIC Educational Resources Information Center

    Roecker, Lee

    2008-01-01

    A two-week laboratory experiment for students in advanced inorganic chemistry is described. Students prepare and characterize a cobalt(III) complex coordinated by a thioether ligand during the first week of the experiment and then study the kinetics of Co-S bond cleavage in basic solution during the second week. The synthetic portion of the…