Simulation of electron-proton coupling with a Monte Carlo method: application to cytochrome c3 using continuum electrostatics.

PubMed Central

Baptista, A M; Martel, P J; Soares, C M

1999-01-01

A new method is presented for simulating the simultaneous binding equilibrium of electrons and protons on protein molecules, which makes it possible to study the full equilibrium thermodynamics of redox and protonation processes, including electron-proton coupling. The simulations using this method reflect directly the pH and electrostatic potential of the environment, thus providing a much closer and realistic connection with experimental parameters than do usual methods. By ignoring the full binding equilibrium, calculations usually overlook the twofold effect that binding fluctuations have on the behavior of redox proteins: first, they affect the energy of the system by creating partially occupied sites; second, they affect its entropy by introducing an additional empty/occupied site disorder (here named occupational entropy). The proposed method is applied to cytochrome c3 of Desulfovibrio vulgaris Hildenborough to study its redox properties and electron-proton coupling (redox-Bohr effect), using a continuum electrostatic method based on the linear Poisson-Boltzmann equation. Unlike previous studies using other methods, the full reduction order of the four hemes at physiological pH is successfully predicted. The sites more strongly involved in the redox-Bohr effect are identified by analysis of their titration curves/surfaces and the shifts of their midpoint redox potentials and pKa values. Site-site couplings are analyzed using statistical correlations, a method much more realistic than the usual analysis based on direct interactions. The site found to be more strongly involved in the redox-Bohr effect is propionate D of heme I, in agreement with previous studies; other likely candidates are His67, the N-terminus, and propionate D of heme IV. Even though the present study is limited to equilibrium conditions, the possible role of binding fluctuations in the concerted transfer of protons and electrons under nonequilibrium conditions is also discussed. The occupational entropy contributions to midpoint redox potentials and pKa values are computed and shown to be significant. PMID:10354425

Selectivity and stoichiometry boosting of beta-cyclodextrin in cationic/anionic surfactant systems: when host-guest equilibrium meets biased aggregation equilibrium.

PubMed

Jiang, Lingxiang; Yu, Caifang; Deng, Manli; Jin, Changwen; Wang, Yilin; Yan, Yun; Huang, Jianbin

2010-02-18

Cationic surfactant/anionic surfactant/beta-CD ternary aqueous systems provide a platform for the coexistence of the host-guest (beta-CD/surfactant) equilibrium and the biased aggregation (monomeric/aggregated surfactants) equilibrium. We report here that the interplay between the two equilibria dominates the systems as follows. (1) The biased aggregation equilibrium imposes an apparent selectivity on the host-guest equilibrium, namely, beta-CD has to always selectively bind the major surfactant (molar fraction > 0.5) even if binding constants of beta-CD to the pair of surfactants are quite similar. (2) In return, the host-guest equilibrium amplifies the bias of the aggregation equilibrium, that is, the selective binding partly removes the major surfactant from the aggregates and leaves the aggregate composition approaching the electroneutral mixing stoichiometry. (3) This composition variation enhances electrostatic attractions between oppositely charged surfactant head groups, thus resulting in less-curved aggregates. In particular, the present apparent host-guest selectivity is of remarkably high values, and the selectivity stems from the bias of the aggregation equilibrium rather than the difference in binding constants. Moreover, beta-CD is defined as a "stoichiometry booster" for the whole class of cationic/anionic surfactant systems, which provides an additional degree of freedom to directly adjust aggregate compositions of the systems. The stoichiometry boosting of the compositions can in turn affect or even determine microstructures and macroproperties of the systems.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

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

PubMed

Drake, Andrew W; Klakamp, Scott L

2007-01-10

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

Equilibrium expert: an add-in to Microsoft Excel for multiple binding equilibrium simulations and parameter estimations.

PubMed

Raguin, Olivier; Gruaz-Guyon, Anne; Barbet, Jacques

2002-11-01

An add-in to Microsoft Excel was developed to simulate multiple binding equilibriums. A partition function, readily written even when the equilibrium is complex, describes the experimental system. It involves the concentrations of the different free molecular species and of the different complexes present in the experiment. As a result, the software is not restricted to a series of predefined experimental setups but can handle a large variety of problems involving up to nine independent molecular species. Binding parameters are estimated by nonlinear least-square fitting of experimental measurements as supplied by the user. The fitting process allows user-defined weighting of the experimental data. The flexibility of the software and the way it may be used to describe common experimental situations and to deal with usual problems such as tracer reactivity or nonspecific binding is demonstrated by a few examples. The software is available free of charge upon request.

Analysis of the difference in color development in the dye-binding method due to the kind of buffer solution.

PubMed

Suzuki, Yuji

2006-06-01

In a dye-binding method using a pH indicator, color development has reportedly been affected by the kind of buffer solution used in the color reagent. This phenomenon was analyzed by using a calculation based on the assumption that the anion of the buffer solution also reacts with protein. Color development decreases with increases in the anion concentration of the buffer solution and in the equilibrium constant of the reaction between the anion and protein. The differences in color development due to the kind of buffer solution can be attributed to differences in the equilibrium constant of the reaction forming the anion-protein complex and to the concentration of the anion between the buffer solutions.

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

PubMed

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

2017-03-01

We report a new analytical method that allows the determination of the magnitude of the equilibrium constant of complexation, K h , of small molecules to C 60 fullerene in aqueous solution. The developed method is based on the up-scaled model of C 60 fullerene-ligand complexation and contains the full set of equations needed to fit titration datasets arising from different experimental methods (UV-Vis spectroscopy, 1 H NMR spectroscopy, diffusion ordered NMR spectroscopy, DLS). The up-scaled model takes into consideration the specificity of C 60 fullerene aggregation in aqueous solution and allows the highly dispersed nature of C 60 fullerene cluster distribution to be accounted for. It also takes into consideration the complexity of fullerene-ligand dynamic equilibrium in solution, formed by various types of self- and hetero-complexes. These features make the suggested method superior to standard Langmuir-type analysis, the approach used to date for obtaining quantitative information on ligand binding with different nanoparticles.

A combined spectroscopic, molecular docking and molecular dynamic simulation study on the interaction of quercetin with β-casein nanoparticles.

PubMed

Mehranfar, Fahimeh; Bordbar, Abdol-Khalegh; Parastar, Hadi

2013-10-05

The interaction of quercetin with β-casein nanoparticle micelle was studied at various temperatures in order to do a complete thermodynamic and molecular analysis on the binding process. The results of fluorescence studies showed the possibility of fluorescence energy transfer between excited tryptophan and quercetin. The determined values of critical transfers distance and the mean distance of ligand from Trp-143 residues in β-casein micelle represents a non-radiative energy transfer mechanism for quenching and the existence of a significant interaction between this flavonoid and β-casein nanoparticle. The equilibrium binding of quercetin with β-casein micelle at different temperatures was studied by using UV-Vis absorption spectroscopy. The chemometric analysis (principal component analysis (PCA) and multivariate curve resolution-alternating least squares (MCR-ALS) methods) on spectrophotometric data revealed the existence of two components in solution (quercetin and β-casein-quercetin complex) and resolved their pure concentration and spectral profiles. This information let us to calculate the equilibrium binding constant at various temperatures and the relevant thermodynamic parameters of interaction (enthalpy, entropy and Gibbs free energy) with low uncertainty. The negative values of entropy and enthalpy changes represent the predominate role of hydrogen binding and van der Waals interactions in the binding process. Docking calculations showed the probable binding site of quercetin is located in the hydrophobic core of β-casein where the quercetin molecule is lined by hydrophobic residues and make five hydrogen bonds and several van der Waals contacts with them. Moreover, molecular dynamic (MD) simulation results suggested that this flavonoid can interact with β-casein, without affecting the secondary structure of β-casein. Simulations, molecular docking and experimental data reciprocally supported each other. Copyright © 2013 Elsevier B.V. All rights reserved.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

PubMed

Delaforge, Elise; Milles, Sigrid; Bouvignies, Guillaume; Bouvier, Denis; Boivin, Stephane; Salvi, Nicola; Maurin, Damien; Martel, Anne; Round, Adam; Lemke, Edward A; Jensen, Malene Ringkjøbing; Hart, Darren J; Blackledge, Martin

2015-12-09

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

PubMed Central

2015-01-01

The crystal structures of channels and transporters reveal the chemical nature of ion-binding sites and, thereby, constrain mechanistic models for their transport processes. However, these structures, in and of themselves, do not reveal equilibrium selectivity or transport preferences, which can be discerned only from various functional assays. In this Review, I explore the relationship between cation transport protein structures, equilibrium binding measurements, and ion transport selectivity. The primary focus is on K+-selective channels and nonselective cation channels because they have been extensively studied both functionally and structurally, but the principles discussed are relevant to other transport proteins and molecules. PMID:26078056

Synthesis of β-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis.

PubMed

Naresh, Kottari; Avaji, Prakash Gouda; Maiti, Krishnagopal; Bharati, Binod K; Syal, Kirtimaan; Chatterji, Dipankar; Jayaraman, Narayanaswamy

2012-04-01

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, β-arabinofuranoside trisaccharide glycolipids constituted with β-(1→2), β-(1→3) and β-(1→2), β-(1→5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying β-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few α-anomeric arabinofuranoside glycolipids showed that glycolipids with β-anomeric linkages were having relatively lower equilibrium binding constants than those with α-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with α-anomeric linkages.

Analytic Approximation of Carbon Condensation Issues in Type ii Supernovae

NASA Astrophysics Data System (ADS)

Clayton, Donald D.

2013-01-01

I present analytic approximations for some issues related to condensation of graphite, TiC, and silicon carbide in oxygen-rich cores of supernovae of Type II. Increased understanding, which mathematical analysis can support, renders researchers more receptive to condensation in O-rich supernova gases. Taking SN 1987A as typical, my first analysis shows why the abundance of CO molecules reaches an early maximum in which free carbon remains more abundant than CO. This analysis clarifies why O-rich gas cannot oxidize C if 56Co radioactivity is as strong as in SN 1987A. My next analysis shows that the CO abundance could be regarded as being in chemical equilibrium if the CO molecule is given an effective binding energy rather than its laboratory dissociation energy. The effective binding energy makes the thermal dissociation rate of CO equal to its radioactive dissociation rate. This preserves possible relevance for the concept of chemical equilibrium. My next analysis shows that the observed abundances of CO and SiO molecules in SN 1987A rule out frequent suggestions that equilibrium condensation of SUNOCONs has occurred following atomic mixing of the He-burning shell with more central zones in such a way as to reproduce roughly the observed spectrum of isotopes in SUNOCONs while preserving C/O > 1. He atoms admixed along with the excess carbon would destroy CO and SiO molecules, leaving their observed abundances unexplained. The final analysis argues that a chemical quasiequilibrium among grains (but not gas) may exist approximately during condensation, so that its computational use is partially justified as a guide to which mineral phases would be stable against reactions with gas. I illustrate this point with quasiequilibrium calculations by Ebel & Grossman that have shown that graphite is stable even when O/C >1 if prominent molecules are justifiably excluded from the calculation of chemical equilibrium.

Anion binding by bambus[6]uril probed in the gas phase and in solution.

PubMed

Révész, Agnes; Schröder, Detlef; Svec, Jan; Wimmerová, Michaela; Sindelar, Vladimir

2011-10-20

Electrospray ionization mass spectrometry (ESI-MS) is used to probe the binding of small anions to the macrocycle of bambus[6]uril. For the halide ions, the experimental patterns suggest F(-) < Cl(-) < Br(-) < I(-), which is consistent with the order of anion binding found in the condensed phase. Parallel equilibrium studies in the condensed phase establish the association constants of halide anions and bambus[6]uril in mixed solvents. A detailed analysis of the mass spectrometric data is used to shed light on the correlations between the binding constants in the condensed phase and the ion abundances observed using ESI-MS. From the analysis it becomes apparent that ESI-MS can indeed represent the situation in solution to some extent, but the sampling in the gas-phase experiment is not 1:1 compared to that in solution.

Carbohydrate binding properties of the stinging nettle (Urtica dioica) rhizome lectin.

PubMed

Shibuya, N; Goldstein, I J; Shafer, J A; Peumans, W J; Broekaert, W F

1986-08-15

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.

Modeling Complex Equilibria in ITC Experiments: Thermodynamic Parameters Estimation for a Three Binding Site Model

PubMed Central

Le, Vu H.; Buscaglia, Robert; Chaires, Jonathan B.; Lewis, Edwin A.

2013-01-01

Isothermal Titration Calorimetry, ITC, is a powerful technique that can be used to estimate a complete set of thermodynamic parameters (e.g. Keq (or ΔG), ΔH, ΔS, and n) for a ligand binding interaction described by a thermodynamic model. Thermodynamic models are constructed by combination of equilibrium constant, mass balance, and charge balance equations for the system under study. Commercial ITC instruments are supplied with software that includes a number of simple interaction models, for example one binding site, two binding sites, sequential sites, and n-independent binding sites. More complex models for example, three or more binding sites, one site with multiple binding mechanisms, linked equilibria, or equilibria involving macromolecular conformational selection through ligand binding need to be developed on a case by case basis by the ITC user. In this paper we provide an algorithm (and a link to our MATLAB program) for the non-linear regression analysis of a multiple binding site model with up to four overlapping binding equilibria. Error analysis demonstrates that fitting ITC data for multiple parameters (e.g. up to nine parameters in the three binding site model) yields thermodynamic parameters with acceptable accuracy. PMID:23262283

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).

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

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

PubMed Central

1982-01-01

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues. PMID:7142954

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

PubMed Central

1980-01-01

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid. PMID:7391812

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

PubMed Central

Kuznetsova, Irina M.; Sulatskaya, Anna I.; Uversky, Vladimir N.; Turoverov, Konstantin K.

2012-01-01

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates. PMID:22383971

Analyzing thioflavin T binding to amyloid fibrils by an equilibrium microdialysis-based technique.

PubMed

Kuznetsova, Irina M; Sulatskaya, Anna I; Uversky, Vladimir N; Turoverov, Konstantin K

2012-01-01

A new approach for the determination of the amyloid fibril - thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

PubMed

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

2016-05-15

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Entrapment of Alpha1-Acid Glycoprotein in High-Performance Affinity Columns for Drug-Protein Binding Studies

PubMed Central

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S.

2015-01-01

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (± 0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. PMID:26627938

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Determination of the absolute binding free energies of HIV-1 protease inhibitors using non-equilibrium molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Ngo, Son Tung; Nguyen, Minh Tung; Nguyen, Minh Tho

2017-05-01

The absolute binding free energy of an inhibitor to HIV-1 Protease (PR) was determined throughout evaluation of the non-bonded interaction energy difference between the two bound and unbound states of the inhibitor and surrounding molecules by the fast pulling of ligand (FPL) process using non-equilibrium molecular dynamics (NEMD) simulations. The calculated free energy difference terms help clarifying the nature of the binding. Theoretical binding affinities are in good correlation with experimental data, with R = 0.89. The paradigm used is able to rank two inhibitors having the maximum difference of ∼1.5 kcal/mol in absolute binding free energies.

Assessment of the binding performance of histamine-imprinted microspheres by frontal analysis capillary electrophoresis.

PubMed

Romano, Edwin F; Quirino, Joselito P; Holdsworth, John L; So, Regina C; Holdsworth, Clovia I

2017-05-01

Frontal analysis capillary electrophoresis was used to evaluate the binding performance of molecularly imprinted microspheres (MIM) toward its template histamine and analogs at pH 7, and compared to the high performance liquid chromatographic method. In both methods, batch binding was employed and the binding parameters were calculated from the measured concentration of unbound amine analytes and afforded comparable histamine equilibrium dissociation constants (K d ∼ 0.4 mM). FACE was easily carried out at shorter binding equilibration time (i.e. 30 min) and without the need to separate the microspheres, circumventing laborious and, in the case of the system under study, inefficient sample filtration. It also allowed for competitive binding studies by virtue of its ability to distinctly separate intact microspheres and all tested amines which could not be resolved in HPLC. K d 's for nonimprinted (control) microspheres (NIM) from FACE and HPLC were also comparable (∼ 0.6 mM) but at higher histamine concentrations, HPLC gave lower histamine binding. This discrepancy was attributed to inefficient filtration of the batch binding samples prior to HPLC analysis resulting in an over-estimation of the concentration of free histamine brought about by the presence of unfiltered histamine-bound microspheres. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein.

PubMed

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E; Fernandez-Alberti, Sebastian

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

NASA Astrophysics Data System (ADS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E.; Fernandez-Alberti, Sebastian

2015-06-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

Impact of mutations on the allosteric conformational equilibrium

PubMed Central

Weinkam, Patrick; Chen, Yao Chi; Pons, Jaume; Sali, Andrej

2012-01-01

Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction. PMID:23228330

Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

PubMed Central

Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

2016-01-01

Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

A Printed Equilibrium Dialysis Device with Integrated Membranes for Improved Binding Affinity Measurements.

PubMed

Pinger, Cody W; Heller, Andrew A; Spence, Dana M

2017-07-18

Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ∼3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system's requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn 2+ and human serum albumin (K d = (5.62 ± 0.93) × 10 -7 M) under physiological conditions that is statistically equal to the constants reported in the literature.

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.

PubMed

Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A

2004-03-26

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

Design and structure of an equilibrium protein folding intermediate: a hint into dynamical regions of proteins.

PubMed

Ayuso-Tejedor, Sara; Angarica, Vladimir Espinosa; Bueno, Marta; Campos, Luis A; Abián, Olga; Bernadó, Pau; Sancho, Javier; Jiménez, M Angeles

2010-07-23

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing.

PubMed

Jankowsky, Eckhard; Harris, Michael E

2017-04-15

To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes. Copyright © 2017 Elsevier Inc. All rights reserved.

The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

PubMed Central

Zheng, Xiliang; Wang, Jin

2015-01-01

We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

Quantitative analysis of small molecule-nucleic acid interactions with a biosensor surface and surface plasmon resonance detection.

PubMed

Liu, Yang; Wilson, W David

2010-01-01

Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented.

Comparative study of the molecularly imprinted polymers prepared by reversible addition-fragmentation chain transfer "bulk" polymerization and traditional radical "bulk" polymerization.

PubMed

Ma, Yue; Pan, Guoqing; Zhang, Ying; Guo, Xianzhi; Zhang, Huiqi

2013-05-01

Bisphenol A (BPA) and propranolol-imprinted polymers have been prepared via both reversible addition-fragmentation chain transfer "bulk" polymerization (RAFTBP) and traditional radical "bulk" polymerization (TRBP) under similar reaction conditions, and their equilibrium binding properties were compared in detail for the first time. The chemical compositions, specific surface areas, equilibrium bindings, and selectivity of the obtained molecularly imprinted polymers (MIPs) were systematically characterized. The experimental results showed that the MIPs with molecular imprinting effects and quite fast binding kinetics could be readily prepared via RAFTBP, but they did not show improved template binding properties in comparison with those prepared via TRBP, which is in sharp contrast to many previous reports. This could be attributed to the heavily interrupted equilibrium between the dormant species and active radicals in the RAFT mechanism because of the occurrence of fast gelation during RAFTBP. The findings presented here strongly demonstrates that the application of controlled radical polymerizations (CRPs) in molecular imprinting does not always benefit the binding properties of the resultant MIPs, which is of significant importance for the rational use of CRPs in generating MIPs with improved properties. Copyright © 2013 John Wiley & Sons, Ltd.

STUDIES OF VERAPAMIL BINDING TO HUMAN SERUM ALBUMIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Chen, Sike; Hage, David S.

2008-01-01

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body. PMID:18980867

Mass-action equilibrium and non-specific interactions in protein binding networks

NASA Astrophysics Data System (ADS)

Maslov, Sergei

2009-03-01

Large-scale protein binding networks serve as a paradigm of complex properties of living cells. These networks are naturally weighted with edges characterized by binding strength and protein-nodes -- by their concentrations. However, the state-of-the-art high-throughput experimental techniques generate just a binary (yes or no) information about individual interactions. As a result, most of the previous research concentrated just on topology of these networks. In a series of recent publications [1-4] my collaborators and I went beyond purely topological studies and calculated the mass-action equilibrium of a genome-wide binding network using experimentally determined protein concentrations, localizations, and reliable binding interactions in baker's yeast. We then studied how this equilibrium responds to large perturbations [1-2] and noise [3] in concentrations of proteins. We demonstrated that the change in the equilibrium concentration of a protein exponentially decays (and sign-alternates) with its network distance away from the perturbed node. This explains why, despite a globally connected topology, individual functional modules in such networks are able to operate fairly independently. In a separate study [4] we quantified the interplay between specific and non-specific binding interactions under crowded conditions inside living cells. We show how the need to limit the waste of resources constrains the number of types and concentrations of proteins that are present at the same time and at the same place in yeast cells. [1] S Maslov, I. Ispolatov, PNAS 104:13655 (2007). [2] S. Maslov, K. Sneppen, I. Ispolatov, New J. of Phys. 9: 273 (2007). [3] K-K. Yan, D. Walker, S. Maslov, PRL accepted (2008). [4] J. Zhang, S. Maslov, and E. I. Shakhnovich, Mol Syst Biol 4, 210 (2008).

Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

PubMed Central

Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

2015-01-01

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

Adsorption of Ag (I) from aqueous solution by waste yeast: kinetic, equilibrium and mechanism studies.

PubMed

Zhao, Yufeng; Wang, Dongfang; Xie, Hezhen; Won, Sung Wook; Cui, Longzhe; Wu, Guiping

2015-01-01

One type of biosorbents, brewer fermentation industry waste yeast, was developed to adsorb the Ag (I) in aqueous solution. The result of FTIR analysis of waste yeast indicated that the ion exchange, chelating and reduction were the main binding mechanisms between the silver ions and the binding sites on the surface of the biomass. Furthermore, TEM, XRD and XPS results suggested that Ag(0) nanoparticles were deposited on the surface of yeast. The kinetic experiments revealed that sorption equilibrium could reach within 60 min, and the removal efficiency of Ag (I) could be still over 93 % when the initial concentration of Ag (I) was below 100 mg/L. Thermodynamic parameters of the adsorption process (ΔG, ΔH and ΔS) identified that the adsorption was a spontaneous and exothermic process. The waste yeast, playing a significant role in the adsorption of the silver ions, is useful to fast adsorb Ag (I) from low concentration.

The binding of aluminum to mugineic acid and related compounds as studied by potentiometric titration.

PubMed

Yoshimura, Etsuro; Kohdr, Hicham; Mori, Satoshi; Hider, Robert C

2011-08-01

The phytosiderophores, mugineic acid (MA) and epi-hydroxymugineic acid (HMA), together with a related compound, nicotianamine (NA), were investigated for their ability to bind Al(III). Potentiometric titration analysis demonstrated that MA and HMA bind Al(III), in contrast to NA which does not under normal physiological conditions. With MA and HMA, in addition to the Al complex (AlL), the protonated (AlLH) and deprotonated (AlLH(-1)) complexes were identified from an analysis of titration curves, where L denotes the phytosiderophore form in which all the carboxylate functions are ionized. The equilibrium formation constants of the Al(III) phytosiderophore complexes are much smaller than those of the corresponding Fe(III) complexes. The higher selectivity of phytosiderophores for Fe(III) over Al(III) facilitates Fe(III) acquisition in alkaline conditions where free Al(III) levels are higher than free Fe(III) levels.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I.

PubMed

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B; Erman, James E

2014-01-03

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0. Copyright © 2013 Elsevier Inc. All rights reserved.

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

PubMed Central

Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B.; Erman, James E.

2013-01-01

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M−1s−1 and 0.34 ± 0.15 s−1, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min−1 at pH 6.0. PMID:24291498

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

PubMed Central

Qian, Yufeng; Johnson, Kenneth A.

2017-01-01

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m−1 s−1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. PMID:28615444

Exploring the interaction of silver nanoparticles with pepsin and its adsorption isotherms and kinetics.

PubMed

Li, Xiangrong; Wang, Kaiwei; Peng, Yanru

2018-04-25

The interaction of nanoparticles (NPs) with proteins is a topic of high relevance for the medical application of nanomaterials. In the study, a comprehensive investigation was performed for the binding properties of silver nanoparticles (AgNPs) to pepsin. The results indicate that the binding of AgNPs to pepsin may be a static quenching mechanism. Thermodynamic analysis reveals that AgNPs binds to pepsin is synergistically driven by enthalpy and entropy, and the major driving forces are hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy shows that AgNPs may induce microenvironmental changes of pepsin. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased. The adsorption of pepsin on AgNPs was analyzed by Langmuir and Freundlich models, suggesting that the equilibrium adsorption data fit well with Freundlich model. The equilibrium adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetic equations. The results indicate that pseudo-second-order kinetic equation better describes the adsorption kinetics. The study provides an accurate and full basic data for clarifying the binding mechanism, adsorption isotherms and kinetic behaviors of AgNPs with pepsin. These fundamental works will provide some new insights into the safe and effective application of AgNPs in biological and medical areas. Copyright © 2018 Elsevier B.V. All rights reserved.

Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance

PubMed Central

Chang, Andrew L.; McKeague, Maureen; Smolke, Christina D.

2015-01-01

Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene-regulatory applications. Here, we describe methods for characterizing aptamer kinetic and equilibrium binding properties using a surface plasmon resonance-based platform. This aptamer characterization platform is broadly useful for studying aptamer–ligand interactions, comparing aptamer properties, screening functional aptamers during in vitro selection processes, and prototyping aptamers for integration into nucleic acid devices. PMID:25432760

The Kinetic Mechanism for Cytochrome P450 Metabolism of Type II Binding Compounds: Evidence Supporting Direct Reduction

PubMed Central

Pearson, Joshua; Dahal, Upendra P.; Rock, Daniel; Peng, Chi-Chi; Schenk, James O.; Joswig-Jones, Carolyn; Jones, Jeffrey P.

2011-01-01

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at subsaturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; 1) Dead-end type II binding, 2) a rapid equilibrium between type I and II binding modes before reduction, and 3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1. PMID:21530484

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawada, Y.; Kawai, R.; McManaway, M.

(3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB);more » estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.« less

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

The structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

PubMed Central

Middendorf, Thomas R.

2017-01-01

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components. PMID:27993951

Increased thyrotropin binding in hyperfunctioning thyroid nodules.

PubMed

Müller-Gärtner, H W; Schneider, C; Bay, V; Tadt, A; Rehpenning, W; de Heer, K; Jessel, M

1987-08-01

The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.

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

PubMed

Montalvo-Acosta, Joel José; Cecchini, Marco

2016-12-01

The physiological role played by protein-ligand recognition has motivated the development of several computational approaches to the ligand binding affinity. Some of them, termed rigorous, have a strong theoretical foundation but involve too much computation to be generally useful. Some others alleviate the computational burden by introducing strong approximations and/or empirical calibrations, which also limit their general use. Most importantly, there is no straightforward correlation between the predictive power and the level of approximation introduced. Here, we present a general framework for the quantitative interpretation of protein-ligand binding based on statistical mechanics. Within this framework, we re-derive self-consistently the fundamental equations of some popular approaches to the binding constant and pinpoint the inherent approximations. Our analysis represents a first step towards the development of variants with optimum accuracy/efficiency ratio for each stage of the drug discovery pipeline. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nonlinear Analyte Concentration Gradients for One-Step Kinetic Analysis Employing Optical Microring Resonators

PubMed Central

Marty, Michael T.; Kuhnline Sloan, Courtney D.; Bailey, Ryan C.; Sligar, Stephen G.

2012-01-01

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics. PMID:22686186

Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators.

PubMed

Marty, Michael T; Sloan, Courtney D Kuhnline; Bailey, Ryan C; Sligar, Stephen G

2012-07-03

Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.

Efficiency of muscle contraction. The chemimechanic equilibrium

NASA Astrophysics Data System (ADS)

Becker, E. W.

1991-10-01

Although muscle contraction is one of the principal themes of biological research, the exact mechanism whereby the chemical free energy of ATP hydrolysis is converted into mechanical work remains elusive. The high thermodynamic efficiency of the process, above all, is difficult to explain on the basis of present theories. A model of the elementary effect in muscle contraction is proposed which aims at high thermodynamic efficiency based on an approximate equilibrium between chemical and mechanical forces throughout the transfer of free energy. The experimental results described in the literature support the assumption that chemimechanic equilibrium is approximated by a free energy transfer system based on the binding of divalent metal ions to the myosin light chains. Muscle contraction demonstrated without light chains is expected to proceed with a considerably lower efficiency. Free energy transfer systems based on the binding of ions to proteins seem to be widespread in the cell. By establishing an approximate chemimechanic equilibrium, they could facilitate biological reactions considerably and save large amounts of free energy. The concept of chemimechanic equilibrium is seen as a supplementation to the concept of chemiosmotic equilibrium introduced for the membrane transport by P. Mitchell.

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

PubMed

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

[18F]CFT [(18F)WIN 35,428], a radioligand to study the dopamine transporter with PET: characterization in human subjects.

PubMed

Laakso, A; Bergman, J; Haaparanta, M; Vilkman, H; Solin, O; Hietala, J

1998-03-01

We have characterized the usage of [18F]CFT (also known as [18F]WIN 35,428) as a radioligand for in vivo studies of human dopamine transporter by PET. CFT was labeled with 18F to a high specific activity, and dynamic PET scans were conducted in healthy volunteers at various time points up to 5 h from [18F]CFT injection. The regional distribution of [18F]CFT uptake correlated well with the known distribution of dopaminergic nerve terminals in the human brain and also with that of other dopamine transporter radioligands. Striatal binding peaked at 225 min after injection and declined thereafter, demonstrating the reversible nature of the binding to the dopamine transporter. Therefore, due to the relatively long half-life of 18F (109.8 min), PET scans with [18F]CFT could easily be conducted during the binding equilibrium, allowing estimation of Bmax/Kd values (i.e., binding potential). Binding potentials for putamen and caudate measured at equilibrium were 4.79+/-0.11 and 4.50+/-0.23, respectively. We were able to also visualize midbrain dopaminergic neurons (substantia nigra) with [18F]CFT in some subjects. In conclusion, the labeling of CFT with 18F allows PET scans to be conducted at binding equilibrium, and therefore a high signal-to-noise ratio and reliable quantification of binding potential can be achieved. With a high resolution 3D PET scanner, the quantification of extrastriatal dopamine transporters should become possible.

Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

PubMed Central

Mukhtasimova, Nuriya; daCosta, Corrie J.B.

2016-01-01

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist–receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy. PMID:27353445

CO 2 Binding Organic Liquids Gas Capture with Polarity Swing Assisted Regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heldebrant, David

This report outlines the comprehensive bench-scale testing of the CO 2-binding organic liquids (CO 2BOLs) solvent platform and its unique Polarity Swing Assisted Regeneration (PSAR). This study outlines all efforts on a candidate CO 2BOL solvent molecule, including solvent synthesis, material characterization, preliminary toxicology studies, and measurement of all physical, thermodynamic and kinetic data, including bench-scale testing. Equilibrium and kinetic models and analysis were made using Aspen Plus™. Preliminary process configurations, a technoeconomic assessment and solvent performance projections for separating CO 2 from a subcritical coal-fired power plant are compared to the U.S. Department of Energy's Case 10 monoethanolamine baseline.

Contacts between the factor TUF and RPG sequences.

PubMed

Vignais, M L; Huet, J; Buhler, J M; Sentenac, A

1990-08-25

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

A long-term stability study of Prussian blue: A quality assessment of water content and cesium binding.

PubMed

Mohammad, Adil; Yang, Yongsheng; Khan, Mansoor A; Faustino, Patrick J

2015-01-25

Prussian blue (PB) is the active pharmaceutical ingredient (API) of Radiogardase, the first approved medical countermeasure for the treatment of radiocesium poisoning in the event of a major radiological incident such as a "dirty bomb" or nuclear attack. The purpose of this study is to assess the long-term stability of Prussian blue drug products (DPs) and APIs under laboratory storage condition by monitoring the loss in water content and the in vitro cesium binding. The water content was measured by thermal gravimetric analysis (TGA). The in-vitro cesium binding study was conducted using a surrogate model to mimic gastric residence and intestinal transport. Free cesium was analyzed using a validated flame atomic emission spectroscopy (AES) method. The binding equilibrium was reached at 24h. The Langmuir isotherm was plotted to calculate the maximum binding capacity (MBC). Comparison of the same PB samples with 2003 data samples, the water content of both APIs and DPs decreased on an average by approximately 12-24%. Consequently, the MBC of cesium was decreased from 358mg/g in 2003 to 265mg/g @ pH 7.5, a decrease of approximately 26%. The binding of cesium is also pH dependent with lowest binding at pH 1.0 and maximum binding at pH 7.5. At pH 7.5, the amount of cesium bound decreased by an average value of 7.9% for APIs and 8.9% for DPs (for 600ppm initial cesium concentration). These findings of water loss, pH dependence and decrease in cesium binding are consistent with our previously published data in 2003. Over last 10 years the stored DPs and APIs of PB have lost about 20% of water which has a negative impact on the PB cesium binding, however PB still meets the FDA specification of >150mg/g at equilibrium. The study is the first quantitative assessment of the long-term stability of PB and directs that proper long-term and short-term storage of PB is required to ensure that it is safe and efficacious at the time of an emergency situation. Published by Elsevier B.V.

Quantification of metabotropic glutamate subtype 5 receptors in the brain by an equilibrium method using 18F-SP203.

PubMed

Kimura, Yasuyuki; Siméon, Fabrice G; Zoghbi, Sami S; Zhang, Yi; Hatazawa, Jun; Pike, Victor W; Innis, Robert B; Fujita, Masahiro

2012-02-01

A new PET ligand, 3-fluoro-5-(2-(2-(18)F-(fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile (18F-SP203) can quantify metabotropic glutamate subtype 5 receptors (mGluR5) in human brain by a bolus injection and kinetic modeling. As an alternative approach to a bolus injection, binding can simply be measured as a ratio of tissue to metabolite-corrected plasma at a single time point under equilibrium conditions achieved by administering the radioligand with a bolus injection followed by a constant infusion. The purpose of this study was to validate the equilibrium method as an alternative to the standard kinetic method for measuring 18F-SP203 binding in the brain. Nine healthy subjects were injected with 18F-SP203 using a bolus plus constant infusion for 300 min. A single ratio of bolus-to-constant infusion (the activity of bolus equaled to that of infusion over 219 min) was applied to all subjects to achieve equilibrium in approximately 120 min. As a measure of ligand binding, we compared total distribution volume (VT) calculated by the equilibrium and kinetic methods in each scan. The equilibrium method calculated VT by the ratio of radioactivity in the brain to the concentration of 18F-SP203 in arterial plasma at 120 min, and the kinetic method calculated VT by a two-tissue compartment model using brain and plasma dynamic data from 0 to 120 min. VT obtained via the equilibrium method was highly correlated with VT obtained via kinetic modeling. Inter-subject variability of VT obtained via the equilibrium method was slightly smaller than VT obtained via the kinetic method. VT obtained via the equilibrium method was ~10% higher than VT obtained via the kinetic method, indicating a small difference between the measurements. Taken together, the results of this study show that using the equilibrium method is an acceptable alternative to the standard kinetic method when using 18F-SP203 to measure mGluR5. Although small differences in the measurements obtained via the equilibrium and kinetic methods exist, both methods consistently measured mGluR5 as indicated by the highly correlated VT values; the equilibrium method was slightly more precise, as indirectly measured by the smaller coefficient of variability across subjects. In addition, when using 18F-SP203, the equilibrium method is more efficient because it requires much less data. Copyright © 2011. Published by Elsevier Inc.

Energy transport pathway in proteins: Insights from non-equilibrium molecular dynamics with elastic network model.

PubMed

Wang, Wei Bu; Liang, Yu; Zhang, Jing; Wu, Yi Dong; Du, Jian Jun; Li, Qi Ming; Zhu, Jian Zhuo; Su, Ji Guo

2018-06-22

Intra-molecular energy transport between distant functional sites plays important roles in allosterically regulating the biochemical activity of proteins. How to identify the specific intra-molecular signaling pathway from protein tertiary structure remains a challenging problem. In the present work, a non-equilibrium dynamics method based on the elastic network model (ENM) was proposed to simulate the energy propagation process and identify the specific signaling pathways within proteins. In this method, a given residue was perturbed and the propagation of energy was simulated by non-equilibrium dynamics in the normal modes space of ENM. After that, the simulation results were transformed from the normal modes space to the Cartesian coordinate space to identify the intra-protein energy transduction pathways. The proposed method was applied to myosin and the third PDZ domain (PDZ3) of PSD-95 as case studies. For myosin, two signaling pathways were identified, which mediate the energy transductions form the nucleotide binding site to the 50 kDa cleft and the converter subdomain, respectively. For PDZ3, one specific signaling pathway was identified, through which the intra-protein energy was transduced from ligand binding site to the distant opposite side of the protein. It is also found that comparing with the commonly used cross-correlation analysis method, the proposed method can identify the anisotropic energy transduction pathways more effectively.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

PubMed

Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-01-01

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Probing receptor-ligand interactions by sedimentation equilibrium

NASA Astrophysics Data System (ADS)

Philo, John S.

1997-05-01

While sedimentation equilibrium is most commonly used to characterize the molecular weight and state of association of single proteins, this technique is also a very powerful tool for probing the interactions between two or more different proteins, and can characterize both the binding stoichiometry and the equilibrium constants. To resolve the complex binding interactions that can occur in such systems, it is crucial to globally fit data from many experiments to a common binding model, including samples made with different mixing ratios and a wide range of total concentration. It is often also essential to constrain the parameters during fitting so that the fits correctly reproduce the molar ratio of proteins used in making each sample. We have applied this methodology to probe mechanisms of receptor activation for a number of hematopoietic receptors and their cognate ligands, using receptor extracellular domains expressed as soluble proteins. Such data can potentially help in the design of improved or new protein therapeutics, as well as in efforts to create small- molecular mimetics of protein hormones through structure- based drug design. Sedimentation equilibrium has shown that stem cell factor, erythropoietin, and granulocyte-colony stimulating factor can each dimerize their respective receptors in solution, but the mechanism of ligand-induced receptor dimerization for these three systems are strikingly different.

Binding Isotherms and Time Courses Readily from Magnetic Resonance.

PubMed

Xu, Jia; Van Doren, Steven R

2016-08-16

Evidence is presented that binding isotherms, simple or biphasic, can be extracted directly from noninterpreted, complex 2D NMR spectra using principal component analysis (PCA) to reveal the largest trend(s) across the series. This approach renders peak picking unnecessary for tracking population changes. In 1:1 binding, the first principal component captures the binding isotherm from NMR-detected titrations in fast, slow, and even intermediate and mixed exchange regimes, as illustrated for phospholigand associations with proteins. Although the sigmoidal shifts and line broadening of intermediate exchange distorts binding isotherms constructed conventionally, applying PCA directly to these spectra along with Pareto scaling overcomes the distortion. Applying PCA to time-domain NMR data also yields binding isotherms from titrations in fast or slow exchange. The algorithm readily extracts from magnetic resonance imaging movie time courses such as breathing and heart rate in chest imaging. Similarly, two-step binding processes detected by NMR are easily captured by principal components 1 and 2. PCA obviates the customary focus on specific peaks or regions of images. Applying it directly to a series of complex data will easily delineate binding isotherms, equilibrium shifts, and time courses of reactions or fluctuations.

NMR Mapping of Protein Conformational Landscapes using Coordinated Behavior of Chemical Shifts upon Ligand Binding

PubMed Central

Cembran, Alessandro; Kim, Jonggul; Gao, Jiali; Veglia, Gianluigi

2014-01-01

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria following linear trajectories of NMR chemical shifts. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues’ response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein’s approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins. PMID:24604024

Thermodynamic versus non-equilibrium stability of palmitic acid monolayers in calcium-enriched sea spray aerosol proxy systems.

PubMed

Wellen Rudd, Bethany A; Vidalis, Andrew S; Allen, Heather C

2018-04-16

Of the major cations in seawater (Na+, Mg2+, Ca2+, K+), Ca2+ is found to be the most enriched in fine sea spray aerosols (SSA). In this work, we investigate the binding of Ca2+ to the carboxylic acid headgroup of palmitic acid (PA), a marine-abundant fatty acid, and the impact such binding has on the stability of PA monolayers in both equilibrium and non-equilibrium systems. A range of Ca2+ conditions from 10 μM to 300 mM was utilized to represent the relative concentration of Ca2+ in high and low relative humidity aerosol environments. The CO2- stretching modes of PA detected by surface-sensitive infrared reflection-absorption spectroscopy (IRRAS) reveal ionic binding motifs of the Ca2+ ion to the carboxylate group with varying degrees of hydration. Surface tensiometry was used to determine the thermodynamic equilibrium spreading pressure (ESP) of PA on the various aqueous CaCl2 subphases. Up to concentrations of 1 mM Ca2+, each system reached equilibrium, and Ca2+:PA surface complexation gave rise to a lower energy state revealed by elevated surface pressures relative to water. We show that PA films are not thermodynamically stable at marine aerosol-relevant Ca2+ concentrations ([Ca2+] ≥ 10 mM). IRRAS and vibrational sum frequency generation (VSFG) spectroscopy were used to investigate the surface presence of PA on high concentration Ca2+ aqueous subphases. Non-equilibrium relaxation (NER) experiments were also conducted and monitored by Brewster angle microscopy (BAM) to determine the effect of the Ca2+ ions on PA stability. At high surface pressures, the relaxation mechanisms of PA varied among the systems and were dependent on Ca2+ concentration.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

PubMed Central

Tahara, Atsuo; Tsukada, Junko; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Tanaka, Akihiro

2000-01-01

[3H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [3H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (Kd) of 0.76 nM and a maximum receptor density (Bmax) of 153 fmol mg−1 protein. The Hill coefficient (nH) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [3H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [3H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu1,6]-oxytocin>AVP= atosiban>d(CH2)5Tyr(Me)AVP>[Thr4,Gly7]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca2+]i increase and hyperplasia. In contrast, the V1A receptor selective antagonist, SR 49059, and the V2 receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca2+]i increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca2+]i increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [3H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca2+]i increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. PMID:10694212

Kinetics and equilibria of cyanide binding to prostaglandin H synthase.

PubMed

MacDonald, I D; Dunford, H B

1989-09-01

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

Fluctuations in Mass-Action Equilibrium of Protein Binding Networks

NASA Astrophysics Data System (ADS)

Yan, Koon-Kiu; Walker, Dylan; Maslov, Sergei

2008-12-01

We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by slow changes in total concentrations of interacting proteins. The second type (spontaneous) is caused by quickly decaying thermodynamic deviations away from equilibrium. We investigate the effects of network connectivity on fluctuations by comparing them to scenarios in which the interacting pair is isolated from the network and analytically derives bounds on fluctuations. Collective effects are shown to sometimes lead to large amplification of spontaneous fluctuations. The strength of both types of fluctuations is positively correlated with the complex connectivity and negatively correlated with complex concentration. Our general findings are illustrated using a curated network of protein interactions and multiprotein complexes in baker’s yeast, with empirical protein concentrations.

In vitro interaction study of retinoic acid isomers with telmisartan and amlodipine by equilibrium dialysis method using UV spectroscopy

NASA Astrophysics Data System (ADS)

Varghese, Susheel John; Johny, Sojimol K.; Paul, David; Ravi, Thengungal Kochupappy

2011-07-01

The in vitro protein binding of retinoic acid isomers (isotretinoin and tretinoin) and the antihypertensive drugs (amlodipine and telmisartan) was studied by equilibrium dialysis method. In this study, free fraction of drugs and the % of binding of drugs in the mixture to bovine serum albumin (BSA) were calculated. The influence of retinoic acid isomers on the % of protein binding of telmisartan and amlodipine at physiological pH (7.4) and temperature (37 ± 0.5 °C) was also evaluated. The in vitro displacement interaction study of drugs telmisartan and amlodipine on retinoic acid isomers and also interaction of retinoic acid isomers on telmisartan and amlodipine were carried out.

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

PubMed Central

Nallamsetty, Sreedevi; Waugh, David S.

2007-01-01

Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its “open” and “closed” conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement. PMID:17964542

Interactions of tea tannins and condensed tannins with proteins.

PubMed

Frazier, Richard A; Deaville, Eddie R; Green, Rebecca J; Stringano, Elisabetta; Willoughby, Ian; Plant, John; Mueller-Harvey, Irene

2010-01-20

Binding parameters for the interactions of four types of tannins: tea catechins, grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins, and sorghum procyanidins (mDP=17), with gelatin and bovine serum albumin (BSA) have been determined from isothermal titration calorimetry data. Equilibrium binding constants determined for the interaction with gelatin were in the range 10(4) to 10(6) M(-1) and in the order: sorghum procyanidins > grape seed proanthocyanidins > mimosa 5-deoxy proanthocyanidins > tea catechins. Interaction with BSA was generally weaker, with equilibrium binding constants of < or =10(3)M(-1) for grape seed proanthocyanidins, mimosa 5-deoxy proanthocyanidins and tea catechins, and 10(4)M(-1) for the sorghum procyanidins. In all cases the interactions with proteins were exothermic and involved multiple binding sites on the protein. The data are discussed in relation to the structures and the known nutritional effects of the condensed tannins.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

The C2'- and C3'-endo equilibrium for AMP molecules bound in the cystathionine-beta-synthase domain.

PubMed

Feng, Na; Qi, Chao; Hou, Yan-Jie; Zhang, Ying; Wang, Da-Cheng; Li, De-Feng

2018-03-04

The equilibrium between C2'- and C3'-endo conformations of nucleotides in solution, as well as their polymers DNA and RNA, has been well studied in previous work. However, this equilibrium of nucleotides in their binding state remains unclear. We observed two AMP molecules, in C3'- and C2'-endo conformations respectively, simultaneously bound to a cystathionine-beta-synthase (CBS) domain dimer of the magnesium and cobalt efflux protein CorC in the crystallographic study. The C2'-endo AMP molecule assumes the higher sugar pucker energy and one more hydrogen bond with the protein than the C3'-endo molecule does. The balance between the high sugar pucker energy and the low binding energy suggests an equilibrium or switch between C2'- and C3'-endo conformations of the bound nucleotides. Our work challenge the previous hypothesis that the ribose of the bound nucleotides would be locked in a fixed conformation. Copyright © 2018 Elsevier Inc. All rights reserved.

Kinetics of heavy metal adsorption and desorption in soil: Developing a unified model based on chemical speciation

NASA Astrophysics Data System (ADS)

Peng, Lanfang; Liu, Paiyu; Feng, Xionghan; Wang, Zimeng; Cheng, Tao; Liang, Yuzhen; Lin, Zhang; Shi, Zhenqing

2018-03-01

Predicting the kinetics of heavy metal adsorption and desorption in soil requires consideration of multiple heterogeneous soil binding sites and variations of reaction chemistry conditions. Although chemical speciation models have been developed for predicting the equilibrium of metal adsorption on soil organic matter (SOM) and important mineral phases (e.g. Fe and Al (hydr)oxides), there is still a lack of modeling tools for predicting the kinetics of metal adsorption and desorption reactions in soil. In this study, we developed a unified model for the kinetics of heavy metal adsorption and desorption in soil based on the equilibrium models WHAM 7 and CD-MUSIC, which specifically consider metal kinetic reactions with multiple binding sites of SOM and soil minerals simultaneously. For each specific binding site, metal adsorption and desorption rate coefficients were constrained by the local equilibrium partition coefficients predicted by WHAM 7 or CD-MUSIC, and, for each metal, the desorption rate coefficients of various binding sites were constrained by their metal binding constants with those sites. The model had only one fitting parameter for each soil binding phase, and all other parameters were derived from WHAM 7 and CD-MUSIC. A stirred-flow method was used to study the kinetics of Cd, Cu, Ni, Pb, and Zn adsorption and desorption in multiple soils under various pH and metal concentrations, and the model successfully reproduced most of the kinetic data. We quantitatively elucidated the significance of different soil components and important soil binding sites during the adsorption and desorption kinetic processes. Our model has provided a theoretical framework to predict metal adsorption and desorption kinetics, which can be further used to predict the dynamic behavior of heavy metals in soil under various natural conditions by coupling other important soil processes.

Comparison of solute-binding properties of plastic materials used as pharmaceutical product containers.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy

2010-01-01

Material/water equilibrium binding constants (E(b)) were determined for 11 organic solutes and 2 plastic materials commonly used in pharmaceutical product containers (plasticized polyvinyl chloride and polyolefin). In general, solute binding by the plasticized polyvinyl chloride material was greater, by nearly an order of magnitude, than the binding by the polyolefin (on an equal weight basis). The utilization of the binding constants to facilitate container compatibility assessments (e.g., drug loss by container binding) for drug-containing products is discussed.

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures

PubMed Central

Singh, Pali P.; Hawthorne, Jenci L.; Davis, Christie A.; Quintero, Omar A.

2016-01-01

Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. PMID:27126922

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

A new graphic plot analysis for determination of neuroreceptor binding in positron emission tomography studies.

PubMed

Ito, Hiroshi; Yokoi, Takashi; Ikoma, Yoko; Shidahara, Miho; Seki, Chie; Naganawa, Mika; Takahashi, Hidehiko; Takano, Harumasa; Kimura, Yuichi; Ichise, Masanori; Suhara, Tetsuya

2010-01-01

In positron emission tomography (PET) studies with radioligands for neuroreceptors, tracer kinetics have been described by the standard two-tissue compartment model that includes the compartments of nondisplaceable binding and specific binding to receptors. In the present study, we have developed a new graphic plot analysis to determine the total distribution volume (V(T)) and nondisplaceable distribution volume (V(ND)) independently, and therefore the binding potential (BP(ND)). In this plot, Y(t) is the ratio of brain tissue activity to time-integrated arterial input function, and X(t) is the ratio of time-integrated brain tissue activity to time-integrated arterial input function. The x-intercept of linear regression of the plots for early phase represents V(ND), and the x-intercept of linear regression of the plots for delayed phase after the equilibrium time represents V(T). BP(ND) can be calculated by BP(ND)=V(T)/V(ND)-1. Dynamic PET scanning with measurement of arterial input function was performed on six healthy men after intravenous rapid bolus injection of [(11)C]FLB457. The plot yielded a curve in regions with specific binding while it yielded a straight line through all plot data in regions with no specific binding. V(ND), V(T), and BP(ND) values calculated by the present method were in good agreement with those by conventional non-linear least-squares fitting procedure. This method can be used to distinguish graphically whether the radioligand binding includes specific binding or not.

Out-of-Equilibrium Dynamics of Colloidal Particles at Interfaces

NASA Astrophysics Data System (ADS)

Wang, Anna

It is widely assumed that when colloidal particles adsorb to a fluid-fluid interface, they reach equilibrium rapidly. Recently, however, Kaz et al. [Nature Materials, 11, 138-142 (2012)] found that a variety of functionalised latex microspheres breaching an aqueous phase-oil interface relax logarithmically with time toward equilibrium. The relaxation is so slow that the time projected for the particles to reach the equilibrium contact angle of 110° is months--far longer than typical experimental timescales. In this thesis, we seek to understand the out-of-equilibrium behaviour of particles near interfaces. Because contact line pinning is likely an extra source of dissipation at interfaces, we start with experiments to elucidate the origins of contact-line pinning and find that polymer hairs on aqueous dispersed polymer particles strongly pin the contact-line. For particles without polymer hairs, nanoscale surface roughness can also pin the contact-line, though with a lower energy. We then extend our digital holography capabilities to track non-spherical particles. We demonstrate that we can track the centre-of-mass of a colloidal spherocylinder to a precision of 35 nm in all three dimensions and its orientation to a precision of 1.5°. Furthermore, the measured translational and rotational diffusion coefficients for the spherocylinders agree with hydrodynamic predictions to within 0.3%. This new functionality enables us to track colloidal ellipsoids and spherocylinders as they breach interfaces. By comparing the adsorption trajectories of the non-spherical particles to what is predicted from energy minimisation, we learn that contact-line pinning affects not just the timescales of breaching, but also the pathway to equilibrium. In fact, a particle's path to equilibrium can have complications even before the particle breaches the interface. Some particles are attracted to the interface, but stay within a few nanometers without ever breaching. We refer to this binding-mode as 'non-capillary binding', and we investigate when this binding mode is present, what causes it, and how interparticle interactions depend on the binding mode. The last few chapters in this thesis are extensions of ideas developed in the first part. We track the run and tumble of E.coli to demonstrate the potential of digital holographic microscopy as an imaging tool for active particles. Taking all of the particle-interface literature into account, we also outline some simple design principles for making particle-stabilised Pickering emulsions.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies unravel the active conformation of apo RORγt nuclear receptor and a common inverse agonism of two diverse classes of RORγt inhibitors.

PubMed

Li, Xiang; Anderson, Marie; Collin, Delphine; Muegge, Ingo; Wan, John; Brennan, Debra; Kugler, Stanley; Terenzio, Donna; Kennedy, Charles; Lin, Siqi; Labadia, Mark E; Cook, Brian; Hughes, Robert; Farrow, Neil A

2017-07-14

The nuclear receptor retinoid acid receptor-related orphan receptor γt (RORγt) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. RORγt has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for RORγt. Here, we report the crystal structures for the ligand-binding domain of RORγt in both apo and ligand-bound states. We show that apo RORγt adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of RORγt in the active state in the absence of a ligand. The structures of ligand-bound RORγt reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of RORγt toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the increased binding of acetaldehyde to red blood cells in alcoholics.

PubMed

Hernández-Muñoz, R; Baraona, E; Blacksberg, I; Lieber, C S

1989-10-01

Using equilibrium dialysis, we found that acetaldehyde, at the levels commonly occurring after ethanol ingestion, did not bind detectably to plasma proteins, but there was significant binding to red blood cells, more in alcoholics than in nonalcoholics. The binding to red blood cells was inhibited by pyridoxal phosphate and N-ethylmaleimide, suggesting adduction to amino and thiol groups. Binding kinetics were consistent with at least two sites. The one with the highest affinity for acetaldehyde corresponded to hemoglobin. Its affinity and Bmax were not changed in alcoholics, but these binding sites accounted for only 44% of the sites available in the red blood cells of alcoholics and 80% of those in controls. Moreover, this binding was not inhibited by N-ethylmaleimide. There was no detectable binding to red cell ghosts. Nonprotein binding was then assessed by changes in NADH produced by the addition of protein-free fractions of the cells to an alcohol dehydrogenase system in equilibrium; this revealed a second binder of lower affinity, larger capacity and with sensitivity to both inhibitors. This binding (possibly due to thiazolidine formation with cysteine) was enhanced in alcoholics, whose red blood cell cysteine content was doubled. Levels of red blood cell cysteine and acetaldehyde remained high for 2 weeks after withdrawal. Because of the prolonged persistence after withdrawal, these changes may provide new markers of alcoholism.

Building New Bridges between In Vitro and In Vivo in Early Drug Discovery: Where Molecular Modeling Meets Systems Biology.

PubMed

Pearlstein, Robert A; McKay, Daniel J J; Hornak, Viktor; Dickson, Callum; Golosov, Andrei; Harrison, Tyler; Velez-Vega, Camilo; Duca, José

2017-01-01

Cellular drug targets exist within networked function-generating systems whose constituent molecular species undergo dynamic interdependent non-equilibrium state transitions in response to specific perturbations (i.e.. inputs). Cellular phenotypic behaviors are manifested through the integrated behaviors of such networks. However, in vitro data are frequently measured and/or interpreted with empirical equilibrium or steady state models (e.g. Hill, Michaelis-Menten, Briggs-Haldane) relevant to isolated target populations. We propose that cells act as analog computers, "solving" sets of coupled "molecular differential equations" (i.e. represented by populations of interacting species)via "integration" of the dynamic state probability distributions among those populations. Disconnects between biochemical and functional/phenotypic assays (cellular/in vivo) may arise with targetcontaining systems that operate far from equilibrium, and/or when coupled contributions (including target-cognate partner binding and drug pharmacokinetics) are neglected in the analysis of biochemical results. The transformation of drug discovery from a trial-and-error endeavor to one based on reliable design criteria depends on improved understanding of the dynamic mechanisms powering cellular function/dysfunction at the systems level. Here, we address the general mechanisms of molecular and cellular function and pharmacological modulation thereof. We outline a first principles theory on the mechanisms by which free energy is stored and transduced into biological function, and by which biological function is modulated by drug-target binding. We propose that cellular function depends on dynamic counter-balanced molecular systems necessitated by the exponential behavior of molecular state transitions under non-equilibrium conditions, including positive versus negative mass action kinetics and solute-induced perturbations to the hydrogen bonds of solvating water versus kT. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

AtSPX1 affects the AtPHR1-DNA-binding equilibrium by binding monomeric AtPHR1 in solution.

PubMed

Qi, Wanjun; Manfield, Iain W; Muench, Stephen P; Baker, Alison

2017-10-23

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼50% of agricultural soils. The transcription factor phosphate starvation response 1 (PHR1) plays a central role in regulating the expression of a subset of phosphate starvation-induced (PSI) genes through binding to a cis -acting DNA element termed P1BS (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX ( S yg1, P ho81, X pr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil domain of AtPHR1 fused to maltose-binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2× P1BS-binding site, suggesting an important role for protein co-operativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA-binding signal from MBPAtdPHR1. These data suggest that, in the Pi-restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA-binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA-binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression. © 2017 The Author(s).

Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

2011-03-01

Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use inmore » combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.« less

New Equilibrium Models of Drug-Receptor Interactions Derived from Target-Mediated Drug Disposition.

PubMed

Peletier, Lambertus A; Gabrielsson, Johan

2018-05-14

In vivo analyses of pharmacological data are traditionally based on a closed system approach not incorporating turnover of target and ligand-target kinetics, but mainly focussing on ligand-target binding properties. This study incorporates information about target and ligand-target kinetics parallel to binding. In a previous paper, steady-state relationships between target- and ligand-target complex versus ligand exposure were derived and a new expression of in vivo potency was derived for a circulating target. This communication is extending the equilibrium relationships and in vivo potency expression for (i) two separate targets competing for one ligand, (ii) two different ligands competing for a single target and (iii) a single ligand-target interaction located in tissue. The derived expressions of the in vivo potencies will be useful both in drug-related discovery projects and mechanistic studies. The equilibrium states of two targets and one ligand may have implications in safety assessment, whilst the equilibrium states of two competing ligands for one target may cast light on when pharmacodynamic drug-drug interactions are important. The proposed equilibrium expressions for a peripherally located target may also be useful for small molecule interactions with extravascularly located targets. Including target turnover, ligand-target complex kinetics and binding properties in expressions of potency and efficacy will improve our understanding of within and between-individual (and across species) variability. The new expressions of potencies highlight the fact that the level of drug-induced target suppression is very much governed by target turnover properties rather than by the target expression level as such.

Differential entry of ricin into malignant and normal rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Decastel, M.; Haentjens, G.; Aubery, M.

1989-02-01

The authors have compared the mechanisms of ricin binding to and entry into Zajdela hepatoma cells (ZHC) and normal rat hepatocytes (HyC). Lactose but not mannan was found to inhibit ricin binding to and toxicity on ZHC and HyC. This finding suggests that ricin binding, entry, and toxicity are expressed only through the galactose binding sites on ZHC and HyC. Nevertheless, the characteristics of ricin binding and its entry pathway appeared to be different in several respects in ZHC and HyC. Scatchard analysis of equilibrium data determined over a wide range of {sup 125}I-labeled ricin concentrations yielded a curvilinear plotmore » for ZHC, while a straight line was obtained for HyC. These results indicate that only ZHC possess high-affinity receptors for ricin. Analysis of ricin toxicity of ZHC and HyC, in the presence of ammonium chloride or after K{sup +}-depletion in both cell types, suggests that the ricin bound to galactose receptors entered through neutral vesicles in ZHC, and through both neutral and acidic vesicles in HyC. The qualitative and quantitative differences found between the process of receptor-mediated endocytosis of ricin in ZHC and HyC might explain the differential sensitivity of the two cell types toward the toxin.« less

In vitro interaction study of retinoic acid isomers with telmisartan and amlodipine by equilibrium dialysis method using UV spectroscopy.

PubMed

Varghese, Susheel John; Johny, Sojimol K; Paul, David; Ravi, Thengungal Kochupappy

2011-07-01

The in vitro protein binding of retinoic acid isomers (isotretinoin and tretinoin) and the antihypertensive drugs (amlodipine and telmisartan) was studied by equilibrium dialysis method. In this study, free fraction of drugs and the % of binding of drugs in the mixture to bovine serum albumin (BSA) were calculated. The influence of retinoic acid isomers on the % of protein binding of telmisartan and amlodipine at physiological pH (7.4) and temperature (37±0.5°C) was also evaluated. The in vitro displacement interaction study of drugs telmisartan and amlodipine on retinoic acid isomers and also interaction of retinoic acid isomers on telmisartan and amlodipine were carried out. Copyright © 2011 Elsevier B.V. All rights reserved.

Non-equilibrium voltage noise generated by ion transport through pores.

PubMed

Frehland, E; Solleder, P

1985-01-01

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicitly for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.

N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia.

PubMed

Welch, M; Todd, D E; Whitehead, N A; McGowan, S J; Bycroft, B W; Salmond, G P

2000-02-15

Quorum sensing via an N-acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR-type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N-(3-oxohexanoyl)-L-homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 microM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross-linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M.

1991-05-01

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand bindingmore » studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.« less

An artificial guanine that binds cytidine through the cooperative interaction of metal coordination and hydrogen bonding.

PubMed

Mancin, Fabrizio; Chin, Jik

2002-09-18

Cd(II) complex of L binds selectively to cytidine in DMSO with an equilibrium constant of 117 M-1 (where LH is 2-aminomethyl-8-hydroxyquinoline). In contrast, the Zn(II) complex of L does not bind appreciably to any of the four nucleobases under the same condition used for the Cd(II) complex.

Detection and characterization of nonspecific, sparsely-populated binding modes in the early stages of complexation

PubMed Central

Cardone, A.; Bornstein, A.; Pant, H. C.; Brady, M.; Sriram, R.; Hassan, S. A.

2015-01-01

A method is proposed to study protein-ligand binding in a system governed by specific and non-specific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultra-weak associations lead instead to broader distributions, a manifestation of non-specific, sparsely-populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (pre-relaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. PMID:25782918

Interaction of the dietary pigment curcumin with hemoglobin: energetics of the complexation.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2014-08-01

Thermodynamics of the interaction of the chemotherapeutic and chemopreventive dietary pigment, curcumin, with hemoglobin was studied by isothermal titration calorimetry. The binding was characterized to be exothermic. At 293.15 K, the equilibrium constant for curcumin-Hb complexation was found to be (4.88 ± 0.06) × 10(5) M(-1). The binding stoichiometry was calculated to be 1.08 ± 0.05, confirming a 1:1 complexation. The binding was driven by a large negative standard molar enthalpy change (ΔH(0) = -118.45 ± 0.05 kJ mol(-1)) and an unfavorable standard molar entropy change (TΔS(0) = -86.53 ± 0.01 kJ mol(-1)) at 293.15 K. Increasing the temperature favoured the binding, and the magnitude of the negative standard molar heat capacity change suggested the involvement of significant hydrophobic forces in the binding process. With increasing salt concentration, the magnitude of the equilibrium constant decreased slightly; and the complexation mostly involved non-polyelectrolytic forces contributing about 92-94% of the standard molar Gibbs energy change. DSC studies revealed that curcumin binding caused a partial unfolding of the protein.

Misuse of thermodynamics in the interpretation of isothermal titration calorimetry data for ligand binding to proteins.

PubMed

Pethica, Brian A

2015-03-01

Isothermal titration calorimetry (ITC) has given a mass of data on the binding of small molecules to proteins and other biopolymers, with particular interest in drug binding to proteins chosen as therapeutic indicators. Interpretation of the enthalpy data usually follows an unsound protocol that uses thermodynamic relations in circumstances where they do not apply. Errors of interpretation include incomplete definitions of ligand binding and equilibrium constants and neglect of the non-ideality of the solutions under study, leading to unreliable estimates of standard free energies and entropies of binding. The mass of reported thermodynamic functions for ligand binding to proteins estimated from ITC enthalpies alone is consequently of uncertain thermodynamic significance and utility. ITC and related experiments to test the protocol assumptions are indicated. A thermodynamic procedure avoiding equilibrium constants or other reaction models and not requiring protein activities is given. The discussion draws attention to the fundamental but neglected relation between the thermodynamic activity and bioactivity of drugs and to the generally unknown thermodynamic status of ligand solutions, which for drugs relates directly to effective therapeutic dosimetry. Copyright © 2014 Elsevier Inc. All rights reserved.

First-principles study of the binding energy between nanostructures and its scaling with system size

NASA Astrophysics Data System (ADS)

Tao, Jianmin; Jiao, Yang; Mo, Yuxiang; Yang, Zeng-Hui; Zhu, Jian-Xin; Hyldgaard, Per; Perdew, John P.

2018-04-01

The equilibrium van der Waals binding energy is an important factor in the design of materials and devices. However, it presents great computational challenges for materials built up from nanostructures. Here we investigate the binding-energy scaling behavior from first-principles calculations. We show that the equilibrium binding energy per atom between identical nanostructures can scale up or down with nanostructure size, but can be parametrized for large N with an analytical formula (in meV/atom), Eb/N =a +b /N +c /N2+d /N3 , where N is the number of atoms in a nanostructure and a , b , c , and d are fitting parameters, depending on the properties of a nanostructure. The formula is consistent with a finite large-size limit of binding energy per atom. We find that there are two competing factors in the determination of the binding energy: Nonadditivities of van der Waals coefficients and center-to-center distance between nanostructures. To decode the detail, the nonadditivity of the static multipole polarizability is investigated from an accurate spherical-shell model. We find that the higher-order multipole polarizability displays ultrastrong intrinsic nonadditivity, no matter if the dipole polarizability is additive or not.

Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance.

PubMed

Asher, C; Chigaev, A; Garty, H

2001-09-07

Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC. Copyright 2001 Academic Press.

Fluorescence stopped-flow study of the interaction of tubulin with the antimitotic drug MDL 27048.

PubMed

Silence, K; D'Hoore, A; Engelborghs, Y; Peyrot, V; Briand, C

1992-11-17

The kinetics of the binding of MDL 27048 to tubulin have been studied by fluorescence stopped flow. The binding is accompanied by a fluorescence increase. The time course can be described by a sum of two exponentials, assumed to be due to the presence of two major tubulin isoforms. The observed rate constants depend in a nonlinear way on the concentration of MDL in pseudo-first-order conditions. This concentration dependence can be described by the presence of a fast equilibrium of low affinity, followed by an isomerization of the initial complex. The dissociation kinetics have been studied by displacement experiments, in which MTC was used as a competitive ligand. The reaction enthalpy change for the first binding equilibrium and the activation energies for the forward and reverse steps of the isomerization were determined from the temperature dependence. This was possible for the two tubulin isotype populations. The kinetics of the binding of MDL to tubulin are slowed down in the presence of 3',4',5'-trimethoxyacetophenone, a fast binding analog of the colchicine A-ring, but are not influenced by the binding of tropolone methyl ether, indicating that the binding site of MDL has the A-subsite in common with colchicine, but not the C-subsite.

Humic Acid Complexation of Th, Hf and Zr in Ligand Competition Experiments: Metal Loading and Ph Effects

NASA Technical Reports Server (NTRS)

Stern, Jennifer C.; Foustoukos, Dionysis I.; Sonke, Jeroen E.; Salters, Vincent J. M.

2014-01-01

The mobility of metals in soils and subsurface aquifers is strongly affected by sorption and complexation with dissolved organic matter, oxyhydroxides, clay minerals, and inorganic ligands. Humic substances (HS) are organic macromolecules with functional groups that have a strong affinity for binding metals, such as actinides. Thorium, often studied as an analog for tetravalent actinides, has also been shown to strongly associate with dissolved and colloidal HS in natural waters. The effects of HS on the mobilization dynamics of actinides are of particular interest in risk assessment of nuclear waste repositories. Here, we present conditional equilibrium binding constants (Kc, MHA) of thorium, hafnium, and zirconium-humic acid complexes from ligand competition experiments using capillary electrophoresis coupled with ICP-MS (CE- ICP-MS). Equilibrium dialysis ligand exchange (EDLE) experiments using size exclusion via a 1000 Damembrane were also performed to validate the CE-ICP-MS analysis. Experiments were performed at pH 3.5-7 with solutions containing one tetravalent metal (Th, Hf, or Zr), Elliot soil humic acid (EHA) or Pahokee peat humic acid (PHA), and EDTA. CE-ICP-MS and EDLE experiments yielded nearly identical binding constants for the metal- humic acid complexes, indicating that both methods are appropriate for examining metal speciation at conditions lower than neutral pH. We find that tetravalent metals form strong complexes with humic acids, with Kc, MHA several orders of magnitude above REE-humic complexes. Experiments were conducted at a range of dissolved HA concentrations to examine the effect of [HA]/[Th] molar ratio on Kc, MHA. At low metal loading conditions (i.e. elevated [HA]/[Th] ratios) the ThHA binding constant reached values that were not affected by the relative abundance of humic acid and thorium. The importance of [HA]/[Th] molar ratios on constraining the equilibrium of MHA complexation is apparent when our estimated Kc, MHA values attained at very low metal loading conditions are compared to existing literature data. Overall, experimental data suggest that the tetravalent transition metal/-actinide-humic acid complexation is important over a wide range of pH values, including mildly acidic conditions, and thus, these complexes should be included in speciation models.

A hierarchical approach to cooperativity in macromolecular and self-assembling binding systems.

PubMed

Garcés, Josep Lluís; Acerenza, Luis; Mizraji, Eduardo; Mas, Francesc

2008-04-01

The study of complex macromolecular binding systems reveals that a high number of states and processes are involved in their mechanism of action, as has become more apparent with the sophistication of the experimental techniques used. The resulting information is often difficult to interpret because of the complexity of the scheme (large size and profuse interactions, including cooperative and self-assembling interactions) and the lack of transparency that this complexity introduces into the interpretation of the indexes traditionally used to describe the binding properties. In particular, cooperative behaviour can be attributed to very different causes, such as direct chemical modification of the binding sites, conformational changes in the whole structure of the macromolecule, aggregation processes between different subunits, etc. In this paper, we propose a novel approach for the analysis of the binding properties of complex macromolecular and self-assembling systems. To quantify the binding behaviour, we use the global association quotient defined as K(c) = [occupied sites]/([free sites] L), L being the free ligand concentration. K(c) can be easily related to other measures of cooperativity (such as the Hill number or the Scatchard plot) and to the free energies involved in the binding processes at each ligand concentration. In a previous work, it was shown that K(c) could be decomposed as an average of equilibrium constants in two ways: intrinsic constants for Adair binding systems and elementary constants for the general case. In this study, we show that these two decompositions are particular cases of a more general expression, where the average is over partial association quotients, associated with subsystems from which the system is composed. We also show that if the system is split into different subsystems according to a binding hierarchy that starts from the lower, microscopic level and ends at the higher, aggregation level, the global association quotient can be decomposed following the hierarchical levels of macromolecular organisation. In this process, the partial association quotients of one level are expressed, in a recursive way, as a function of the partial quotients of the level that is immediately below, until the microscopic level is reached. As a result, the binding properties of very complex macromolecular systems can be analysed in detail, making the mechanistic explanation of their behaviour transparent. In addition, our approach provides a model-independent interpretation of the intrinsic equilibrium constants in terms of the elementary ones.

Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

PubMed

Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

2016-12-15

Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spectroscopic and calorimetric studies on the interaction between PAMAM G4-OH and 5-fluorouracil in aqueous solutions

NASA Astrophysics Data System (ADS)

Buczkowski, Adam; Urbaniak, Pawel; Piekarski, Henryk; Palecz, Bartlomiej

2017-01-01

The results of spectroscopic measurements (an increase in solubility, equilibrium dialysis, 1H NMR titration) and calorimetric measurements (isothermal titration ITC) indicate spontaneous (ΔG < 0) binding of 5-fluorouracil molecules by PAMAM G4-OH dendrimer with terminal hydroxyl groups in an aqueous solution. PAMAM G4-OH dendrimer bonds about n = 8 ± 1 molecules of the drug with an equilibrium constant of K = 70 ± 10. The process of saturating the dendrimer active sites by the drug molecules is exothermal (ΔH < 0) and is accompanied by an advantageous change in entropy (ΔS > 0). The parameters of binding 5-fluorouracil by PAMAM G4-OH dendrimer were compared with those of binding this drug by the macromolecules of PAMAM G3-OH and G5-OH.

Equilibrium structure and atomic vibrations of Nin clusters

NASA Astrophysics Data System (ADS)

Borisova, Svetlana D.; Rusina, Galina G.

2017-12-01

The equilibrium bond lengths and binding energy, second differences in energy and vibrational frequencies of free clusters Nin (2 ≤ n ≤ 20) were calculated with the use of the interaction potential obtained in the tight-binding approximation (TBA). The results show that the minimum vibration frequency plays a significant role in the evaluation of the dynamic stability of the clusters. A nonmonotonic dependence of the minimum vibration frequency of clusters on their size and the extreme values for the number of atoms in a cluster n = 4, 6, 13, and 19 are demonstrated. This result agrees with the theoretical and experimental data on stable structures of small metallic clusters.

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

PubMed

Wang, J; Velotta, J B; McDonough, A A; Farley, R A

2001-10-01

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Van’t Hoff global analyses of variable temperature isothermal titration calorimetry data

PubMed Central

Freiburger, Lee A.; Auclair, Karine; Mittermaier, Anthony K.

2016-01-01

Isothermal titration calorimetry (ITC) can provide detailed information on the thermodynamics of biomolecular interactions in the form of equilibrium constants, KA, and enthalpy changes, ΔHA. A powerful application of this technique involves analyzing the temperature dependences of ITC-derived KA and ΔHA values to gain insight into thermodynamic linkage between binding and additional equilibria, such as protein folding. We recently developed a general method for global analysis of variable temperature ITC data that significantly improves the accuracy of extracted thermodynamic parameters and requires no prior knowledge of the coupled equilibria. Here we report detailed validation of this method using Monte Carlo simulations and an application to study coupled folding and binding in an aminoglycoside acetyltransferase enzyme. PMID:28018008

Mechanical measurement of hydrogen bonded host-guest systems under non-equilibrium, near-physiological conditions.

PubMed

Naranjo, Teresa; Cerrón, Fernando; Nieto-Ortega, Belén; Latorre, Alfonso; Somoza, Álvaro; Ibarra, Borja; Pérez, Emilio M

2017-09-01

Decades after the birth of supramolecular chemistry, there are many techniques to measure noncovalent interactions, such as hydrogen bonding, under equilibrium conditions. As ensembles of molecules rapidly lose coherence, we cannot extrapolate bulk data to single-molecule events under non-equilibrium conditions, more relevant to the dynamics of biological systems. We present a new method that exploits the high force resolution of optical tweezers to measure at the single molecule level the mechanical strength of a hydrogen bonded host-guest pair out of equilibrium and under near-physiological conditions. We utilize a DNA reporter to unambiguously isolate single binding events. The Hamilton receptor-cyanuric acid host-guest system is used as a test bed. The force required to dissociate the host-guest system is ∼17 pN and increases with the pulling rate as expected for a system under non-equilibrium conditions. Blocking one of the hydrogen bonding sites results in a significant decrease of the force-to-break by 1-2 pN, pointing out the ability of the method to resolve subtle changes in the mechanical strength of the binding due to the individual H-bonding components. We believe the method will prove to be a versatile tool to address important questions in supramolecular chemistry.

Direct Determination of Vibrational Density of States Change on Ligand Binding to a Protein

NASA Astrophysics Data System (ADS)

Balog, Erika; Becker, Torsten; Oettl, Martin; Lechner, Ruep; Daniel, Roy; Finney, John; Smith, Jeremy C.

2004-07-01

The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium.

DNA binding of a proflavine derivative bearing a platinum hanging residue.

PubMed

Biagini, Silvia; Bianchi, Antonio; Biver, Tarita; Boggioni, Alessia; Nikolayenko, Igor V; Secco, Fernando; Venturini, Marcella

2011-04-01

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I=0.1M and 25°C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides. Copyright © 2010 Elsevier Inc. All rights reserved.

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

Assessing the potential of atomistic molecular dynamics simulations to probe reversible protein-protein recognition and binding

PubMed Central

Abriata, Luciano A.; Dal Peraro, Matteo

2015-01-01

Protein-protein recognition and binding are governed by diffusion, noncovalent forces and conformational flexibility, entangled in a way that only molecular dynamics simulations can dissect at high resolution. Here we exploited ubiquitin’s noncovalent dimerization equilibrium to assess the potential of atomistic simulations to reproduce reversible protein-protein binding, by running submicrosecond simulations of systems with multiple copies of the protein at millimolar concentrations. The simulations essentially fail because they lead to aggregates, yet they reproduce some specificity in the binding interfaces as observed in known covalent and noncovalent ubiquitin dimers. Following similar observations in literature we hint at electrostatics and water descriptions as the main liable force field elements, and propose that their optimization should consider observables relevant to multi-protein systems and unfolded proteins. Within limitations, analysis of binding events suggests salient features of protein-protein recognition and binding, to be retested with improved force fields. Among them, that specific configurations of relative direction and orientation seem to trigger fast binding of two molecules, even over 50 Å distances; that conformational selection can take place within surface-to-surface distances of 10 to 40 Å i.e. well before actual intermolecular contact; and that establishment of contacts between molecules further locks their conformations and relative orientations. PMID:26023027

Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fuchs, G.; Mobassaleh, M.; Donohue-Rolfe, A.

This study examined the binding of purified /sup 125/I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4/sup 0/C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4/sup 0/C. Binding was inversely related to the temperature of incubation. A total of 80% ofmore » the labeled toxin binding at 4/sup 0/C dissociated from MVM when the temperature was raised to 37/sup 0/C, but reassociated when the temperature was again brought to 4/sup 0/C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.« less

Striatal dopamine transporter binding for predicting the development of delayed neuropsychological sequelae in suicide attempters by carbon monoxide poisoning: A SPECT study.

PubMed

Yang, Kai-Chun; Ku, Hsiao-Lun; Wu, Chia-Liang; Wang, Shyh-Jen; Yang, Chen-Chang; Deng, Jou-Fang; Lee, Ming-Been; Chou, Yuan-Hwa

2011-12-30

Carbon monoxide poisoning (COP) after charcoal burning results in delayed neuropsychological sequelae (DNS), which show clinical resemblance to Parkinson's disease, without adequate predictors at present. This study examined the role of dopamine transporter (DAT) binding for the prediction of DNS. Twenty-seven suicide attempters with COP were recruited. Seven of them developed DNS, while the remainder did not. The striatal DAT binding was measured by single photon emission computed tomography with (99m)Tc-TRODAT. The specific uptake ratio was derived based on a ratio equilibrium model. Using a logistic regression model, multiple clinical variables were examined as potential predictors for DNS. COP patients with DNS had a lower binding on left striatal DAT binding than patients without DNS. Logistic regression analysis showed that a combination of initial loss of consciousness and lower left striatal DAT binding predicted the development of DNS. Our data indicate that the left striatal DAT binding could help to predict the development of DNS. This finding not only demonstrates the feasibility of brain imaging techniques for predicting the development of DNS but will also help clinicians to improve the quality of care for COP patients. 2011 Elsevier Ireland Ltd. All rights reserved.

Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

PubMed Central

Almaqwashi, Ali A.; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C.

2016-01-01

There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

PubMed

Tahara, A; Tsukada, J; Tomura, Y; Wada, K i; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Tanaka, A

2000-01-01

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139

Thermodynamic analysis of sol-gel transition of gelatin in terms of water activity in various solutions.

PubMed

Miyawaki, Osato; Omote, Chiaki; Matsuhira, Keiko

2015-12-01

Sol-gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol-gel transition temperature, Tt , was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From Tt and the melting enthalpy, ΔHsol , the equilibrium sol-to-gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman-Tanford equation, which describes the sol-to-gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol-gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol-gel transition. The general solution for the free energy for gel-stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc.

Equilibrium binding behavior of magnesium to wall teichoic acid.

PubMed

Thomas, Kieth J; Rice, Charles V

2015-10-01

Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant. Copyright © 2015 Elsevier B.V. All rights reserved.

Studies of Single Biomolecules, DNA Conformational Dynamics, and Protein Binding

DTIC Science & Technology

2008-07-11

Nucleotide Base pairs Hydrogen bonds FIG. 1: Ladder structure of DNA showing the Watson - Crick bonding of the bases A, T, G, and C which are suspended by a...protected against unwanted action of chemicals and proteins. The three-dimensional structure of DNA is the famed Watson - Crick double-helix, the equilibrium...quantitative analysis [88]. [1] A. Kornberg and T. A. Baker, DNA Replication (W. H. Freeman, New York, 1992). [2] J. D. Watson and F. H. C. Crick

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

PubMed Central

Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.

2015-01-01

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is “balanced,” as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability. PMID:26351676

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

PubMed

Sorci, Mirco; Dassa, Bareket; Liu, Hongwei; Anand, Gaurav; Dutta, Amit K; Pietrokovski, Shmuel; Belfort, Marlene; Belfort, Georges

2013-06-18

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

Volumetrically Derived Thermodynamic Profile of Interactions of Urea with a Native Protein.

PubMed

Son, Ikbae; Chalikian, Tigran V

2016-11-29

We report the first experimental characterization of the full thermodynamic profile for binding of urea to a native protein. We measured the volumetric parameters of lysozyme at pH 7.0 as a function of urea within a temperature range of 18-45 °C. At neutral pH, lysozyme retains its native conformation between 0 and 8 M urea over the entire range of temperatures studied. Consequently, our measured volumetric properties reflect solely the interactions of urea with the native protein and do not involve contributions from urea-induced conformational transitions. We analyzed our data within the framework of a statistical thermodynamic analytical model in which urea-protein interactions are viewed as solvent exchange in the vicinity of the protein. The analysis produced the equilibrium constant, k, for an elementary reaction of urea-protein binding with a change in standard state free energy (ΔG° = -RT ln k) at each experimental temperature. We used the van't Hoff equation to compute from the temperature dependence of the equilibrium constant, k, changes in enthalpy, ΔH°, and entropy, ΔS°, accompanying binding. The thermodynamic profile of urea-protein interactions, in conjunction with published molecular dynamics simulation results, is consistent with the picture in which urea molecules, being underhydrated in the bulk, form strong, enthalpically favorable interactions with the surface protein groups while paying a high entropic price. We discuss ramifications of our results for providing insights into the combined effects of urea, temperature, and pressure on the conformational preferences of proteins.

The investigation of the binding of 6-mercaptopurine to site I on human serum albumin.

PubMed

Sochacka, Jolanta; Baran, Wojciech

2012-12-01

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.

Lead-binding capacity of calcium pectates with different molecular weight.

PubMed

Khotimchenko, Maksim; Makarova, Ksenia; Khozhaenko, Elena; Kovalev, Valeri

2017-04-01

Nowadays, heavy metal contamination of environment is considered as a serious threat to public health because of toxicity of these pollutants and the lack of effective materials with metal-binding properties. Some biopolymers such as pectins were proposed for removal of metal ions from industrial water disposals. Chemical structure of pectins is quite variable and substantially affects their metal binding properties. In this work, relationship between molecular weight and Pb(II)-binding capacity of calcium pectates was investigated in a batch sorption system. The results showed that all pectate samples are able to form complexes with Pb(II) ions. The effects of contact time, pH of the media and equilibrium metal concentration on metal-binding process were tested in experiments. The equilibrium time min required for uptake of Pb(II) by pectate compounds was found to be 60min. Langmuir and Freundlich models were applied for description of interactions between pectates and metal ions. Binding capacity of low molecular pectate was highest among all the samples tested. Langmuir model was figured out to be the best fit within the whole range of pH values. These results demonstrate that calcium pectate with low molecular weight is more promising agent for elimination of Pb(II) ions from contaminated wastewaters. Copyright © 2017 Elsevier B.V. All rights reserved.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

PubMed Central

Gleitsman, Kristin R.

2014-01-01

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

Oxytocin binding sites in bovine mammary tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Xin.

1989-01-01

Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressinmore » binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.« less

Atrazine Molecular Imprinted Polymers: Comparative Analysis by Far-Infrared and Ultraviolet Induced Polymerization

PubMed Central

Chen, Jun; Bai, Lian-Yang; Liu, Kun-Feng; Liu, Run-Qiang; Zhang, Yu-Ping

2014-01-01

Atrazine molecular imprinted polymers (MIPs) were comparatively synthesized using identical polymer formulation by far-infrared (FIR) radiation and ultraviolet (UV)-induced polymerization, respectively. Equilibrium binding experiments were carried out with the prepared MIPs; the results showed that MIPuv possessed specific binding to atrazine compared with their MIPFIR radiation counterparts. Scatchard plot’s of both MIPs indicated that the affinities of the binding sites in MIPs are heterogeneous and can be approximated by two dissociation-constants corresponding to the high-and low-affinity binding sites. Moreover, several common pesticides including atrazine, cyromazine, metamitron, simazine, ametryn, terbutryn were tested to determine their specificity, similar imprinting factor (IF) and different selectivity index (SI) for both MIPs. Physical characterization of the polymers revealed that the different polymerization methods led to slight differences in polymer structures and performance by scanning electron microscope (SEM), Fourier transform infrared absorption (FT-IR), and mercury analyzer (MA). Finally, both MIPs were used as selective sorbents for solid phase extraction (SPE) of atrazine from lake water, followed by high performance liquid chromatography (HPLC) analysis. Compared with commercial C18 SPE sorbent (86.4%–94.8%), higher recoveries of atrazine in spiked lake water were obtained in the range of 90.1%–97.1% and 94.4%–101.9%, for both MIPs, respectively. PMID:24398982

Understanding the impact of Fc glycosylation on its conformational changes by molecular dynamics simulations and bioinformatics.

PubMed

Zhang, Yubo

2015-12-01

N-linked glycosylation of Fc at N297 plays an important role in its effector function, aberrance of which would cause disease pathogenesis. Here, we performed all-atom molecular dynamics simulations to explore the effects of Fc glycosylation on its dynamics behaviors. Firstly, equilibrium simulations suggested that Fc deglycosylation was able to induce residual flexibility in its CH2 domain. Besides, the free energy landscape revealed three minimum energy wells in deglycosylated Fc, representing its "open", "semi-closed" and "closed" states. However, we could only observe the "open" state of glycosylated Fc. Supportively, principal component analysis emphasized the prominent motion of delyclosylated Fc and dynamically depicted how it changed from the "open" state to its "closed" state. Secondly, we studied the recognition mechanism of the Fc binding to its partners. Energy decomposition analysis identified key residues of Fc to recognize its two partners P13 and P34. Evidently, electrostatic potential surfaces showed that electrostatic attraction helped to stabilize the interaction between Fc and its partners. Also, relative binding free energies explained different binding affinities in Fc-P13 and Fc-P34. Collectively, these results together provided the structural basis for understanding conformational changes of deglycosylated Fc and the recognition mechanism of the Fc binding to its partners.

Quantitative in vivo receptor binding. III. Tracer kinetic modeling of muscarinic cholinergic receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.

1985-10-01

A tracer kinetic method is developed for the in vivo estimation of high-affinity radioligand binding to central nervous system receptors. Ligand is considered to exist in three brain pools corresponding to free, nonspecifically bound, and specifically bound tracer. These environments, in addition to that of intravascular tracer, are interrelated by a compartmental model of in vivo ligand distribution. A mathematical description of the model is derived, which allows determination of regional blood-brain barrier permeability, nonspecific binding, the rate of receptor-ligand association, and the rate of dissociation of bound ligand, from the time courses of arterial blood and tissue tracer concentrations.more » The term ''free receptor density'' is introduced to describe the receptor population measured by this method. The technique is applied to the in vivo determination of regional muscarinic acetylcholine receptors in the rat, with the use of (TH)scopolamine. Kinetic estimates of free muscarinic receptor density are in general agreement with binding capacities obtained from previous in vivo and in vitro equilibrium binding studies. In the striatum, however, kinetic estimates of free receptor density are less than those in the neocortex--a reversal of the rank ordering of these regions derived from equilibrium determinations. A simplified model is presented that is applicable to tracers that do not readily dissociate from specific binding sites during the experimental period.« less

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

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

ERIC Educational Resources Information Center

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

2012-01-01

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

Adsorption equilibrium and kinetics of monomer-dimer monoclonal antibody mixtures on a cation exchange resin.

PubMed

Reck, Jason M; Pabst, Timothy M; Hunter, Alan K; Wang, Xiangyang; Carta, Giorgio

2015-07-10

Adsorption equilibrium and kinetics are determined for a monoclonal antibody (mAb) monomer and dimer species, individually and in mixtures, on a macroporous cation exchange resin both under the dilute limit of salt gradient elution chromatography and at high protein loads and low salt based on batch adsorption equilibrium and confocal laser scanning microscopy (CLSM) experiments. In the dilute limit and weak binding conditions, the dimer/monomer selectivity in 10mM phosphate at pH 7 varies between 8.7 and 2.3 decreasing with salt concentration in the range of 170-230mM NaCl. At high protein loads and strong binding conditions (0-60mM NaCl), the selectivity in the same buffer is near unity with no NaCl added, but increases gradually with salt concentration reaching high values between 2 and 15 with 60mM added NaCl. For these conditions, the two-component adsorption kinetics is controlled by pore diffusion and is predicted approximately by a dual shrinking core model using parameters based on single component equilibrium and kinetics measurements. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-02-16

Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

Characterization of autoantibodies to vasoactive intestinal peptide in asthma.

PubMed

Paul, S; Said, S I; Thompson, A B; Volle, D J; Agrawal, D K; Foda, H; de la Rocha, S

1989-07-01

Vasoactive intestinal peptide (VIP) is a potent relaxant of the airway smooth muscle. In this study, VIP-binding autoantibodies were observed in the plasma of 18% asthma patients and 16% healthy subjects. Immunoprecipitation studies and chromatography on DEAE-cellulose and immobilized protein G indicated that the plasma VIP-binding activity was largely due to IgG antibodies. Saturation analysis of VIP binding by the plasmas suggested the presence of one or two classes of autoantibodies, distinguished by their apparent equilibrium affinity constants (Ka). The autoantibodies from asthma patients exhibited a larger VIP-binding affinity compared to those from healthy subjects (Ka 7.8 x 10(9) M-1 and 0.13 x 10(9) M-1, respectively; P less than 0.005). The antibodies were specific for VIP, judged by their poor reaction with peptides bearing partial sequence homology with VIP (peptide histidine isoleucine, growth hormone releasing factor and secretin). IgG prepared from the plasma of an antibody-positive asthma patient inhibited the saturable binding of 125I-VIP by receptors in guinea pig lung membranes (by 39-59%; P less than 0.001). These observations are consistent with a role for the VIP autoantibodies in the airway hyperresponsiveness of asthma.

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps.

PubMed

Bradley, A J; Stoddart, D M

1992-01-01

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Shea, Michael E.; Makhatadze, George I.; Barquera, Blanca

2011-01-01

The Na+-translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na+-NQR enables pumping of Li+, as well as Na+ across the membrane. We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na+-NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with 22Na+ show that, in both its oxidized and reduced states, Na+-NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states. PMID:21652714

Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase

NASA Astrophysics Data System (ADS)

Tran, Diem-Trang T.; Le, Ly T.; Truong, Thanh N.

2013-08-01

Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giannopoulos, G.; Jackson, K.; Kredentser, J.

The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

PubMed Central

Monine, Michael I.; Posner, Richard G.; Savage, Paul B.; Faeder, James R.; Hlavacek, William S.

2010-01-01

Abstract We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-FcεRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important. PMID:20085718

Exploring the stability of ligand binding modes to proteins by molecular dynamics simulations.

PubMed

Liu, Kai; Watanabe, Etsurou; Kokubo, Hironori

2017-02-01

The binding mode prediction is of great importance to structure-based drug design. The discrimination of various binding poses of ligand generated by docking is a great challenge not only to docking score functions but also to the relatively expensive free energy calculation methods. Here we systematically analyzed the stability of various ligand poses under molecular dynamics (MD) simulation. First, a data set of 120 complexes was built based on the typical physicochemical properties of drug-like ligands. Three potential binding poses (one correct pose and two decoys) were selected for each ligand from self-docking in addition to the experimental pose. Then, five independent MD simulations for each pose were performed with different initial velocities for the statistical analysis. Finally, the stabilities of ligand poses under MD were evaluated and compared with the native one from crystal structure. We found that about 94% of the native poses were maintained stable during the simulations, which suggests that MD simulations are accurate enough to judge most experimental binding poses as stable properly. Interestingly, incorrect decoy poses were maintained much less and 38-44% of decoys could be excluded just by performing equilibrium MD simulations, though 56-62% of decoys were stable. The computationally-heavy binding free energy calculation can be performed only for these survived poses.

Mussel-inspired histidine-based transient network metal coordination hydrogels

PubMed Central

Fullenkamp, Dominic E.; He, Lihong; Barrett, Devin G.; Burghardt, Wesley R.; Messersmith, Phillip B.

2013-01-01

Transient network hydrogels cross-linked through histidine-divalent cation coordination bonds were studied by conventional rheologic methods using histidine-modified star poly(ethylene glycol) (PEG) polymers. These materials were inspired by the mussel, which is thought to use histidine-metal coordination bonds to impart self-healing properties in the mussel byssal thread. Hydrogel viscoelastic mechanical properties were studied as a function of metal, pH, concentration, and ionic strength. The equilibrium metal-binding constants were determined by dilute solution potentiometric titration of monofunctional histidine-modified methoxy-PEG and were found to be consistent with binding constants of small molecule analogs previously studied. pH-dependent speciation curves were then calculated using the equilibrium constants determined by potentiometric titration, providing insight into the pH dependence of histidine-metal ion coordination and guiding the design of metal coordination hydrogels. Gel relaxation dynamics were found to be uncorrelated with the equilibrium constants measured, but were correlated to the expected coordination bond dissociation rate constants. PMID:23441102

Adsorption of Pb(II) ions onto biomass from Trifolium resupinatum: equilibrium and kinetic studies

NASA Astrophysics Data System (ADS)

Athar, Makshoof; Farooq, Umar; Aslam, Muhammad; Salman, M.

2013-09-01

The present study provides information about the binding of Pb(II) ions on an eco-friendly and easily available biodegradable biomass Trifolium resupinatum. The powdered biomass was characterized by FTIR, potentiometric titration and surface area analyses. The FTIR spectrum showed the presence of hydroxyl, carbonyl and amino functional groups and Pb(II) ions bound with the oxygen- and nitrogen-containing sites (hydroxyl and amino groups). The acidic groups were also confirmed by titrations. Effects of various environmental parameters (time, pH and concentration) have been studied. The biosorption process achieved equilibrium in a very short period of time (25 min). Non-linear approach for Langmuir and Freundlich models was used to study equilibrium process and root mean-square error was used as an indicator to decide the fitness of the mathematical model. The biosorption process was found to follow pseudo-second-order kinetics and was very fast. Thus, the biomass can be cost-effectively used for the binding of Pb(II) ions from aqueous solutions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strittmatter, S.M.; Snyder, S.H.

We demonstrate that (3H)captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. (3H)Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of (3H)captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of (3H)captopril binding to purified ACE reveals a Bmax of 6 nmol/mgmore » of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of (3H)captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both (3H) captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for (3H)captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2-fold, whereas binding activity was decreased by 70%.« less

Protein associations and analytical ultracentrifugation

NASA Astrophysics Data System (ADS)

Laue, Tom

2010-03-01

Analytical ultracentrifugation (AUC) is a first principle method for characterizing the thermodynamics of macromolecules in solution. Since AUC directly assesses mass, it is particularly useful for characterizing both reversible and irreversible binding interactions between macromolecules. The principle measurement in AUC is the concentration as a function of radial position, which may be provided by either absorbance, interference or fluorescence detection. Each of these three different detectors may be used to characterize protein associations using either sedimentation equilibrium or sedimentation velocity analysis. Examples will be shown for characterizing irreversible (aggregate) formation, high-accuracy reversible association analysis, and the detection of protein interactions in complex and concentrated fluids (e.g. serum, cell cytosol).

Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

PubMed Central

Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

2015-01-01

Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin. PMID:26246073

Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin.

PubMed

Perizzolo, K E; Sullivan, S; Waugh, D F

1985-03-01

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.

ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

2010-01-01

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

Extracellular polymeric substances facilitate the biosorption of phenanthrene on cyanobacteria Microcystis aeruginosa.

PubMed

Bai, Leilei; Xu, Huacheng; Wang, Changhui; Deng, Jiancai; Jiang, Helong

2016-11-01

Phytoplankton-derived extracellular polymeric substances (EPS) are of vital importance for the biogeochemical cycles of hydrophobic organic pollutants in lake ecosystems. In this study, roles of loosely-bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) in biosorption of phenanthrene (PHE) on a typical cyanobacteria Microcystis aeruginosa were investigated. The results showed that the biosorption of PHE on M. aeruginosa cell varied lasted 24 h, while the binding of PHE to LB-EPS and TB-EPS reached equilibrium within less than 2 h. The equilibrium biosorption capacities of M. aeruginosa cell, LB-EPS and TB-EPS were 6.78, 12.31, and 9.47 μg mg(-1), respectively, indicating that the binding of PHE to EPS was a considerable process involved in biosorption. Fluorescence quenching titration revealed that increasing temperature induced more binding sites in EPS for PHE and the binding process was driven by electrostatic force and hydrophobic interactions. Interestingly, dynamic and static quenching processes occurred simultaneously for the binding of PHE to protein-like substances in EPS, whereas the binding of PHE to humic-like substances belonged to static quenching. The relatively higher contents of proteins in LB-EPS produced a stronger binding capacity of PHE. Overall, the interactions between hydrophobic organic pollutants and cyanobacterial EPS are favorable to the bioaccumulation of hydrophobic organic pollutants in cyanobacteria and facilitate the regulatory function of cyanobacterial biomass as a biological pump. Copyright © 2016 Elsevier Ltd. All rights reserved.

Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

PubMed

Mouw, M; Pintel, D J

1998-11-10

GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

PubMed Central

Lapelosa, Mauro; Gallicchio, Emilio; Levy, Ronald M.

2011-01-01

The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations. PMID:22368530

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors.

PubMed Central

Alexander, M D; Andrews, J A; Leslie, R G; Wood, N J

1978-01-01

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2. PMID:680795

Kinetics of Cation and Oxyanion Adsorption and Desorption on Ferrihydrite: Roles of Ferrihydrite Binding Sites and a Unified Model.

PubMed

Tian, Lei; Shi, Zhenqing; Lu, Yang; Dohnalkova, Alice C; Lin, Zhang; Dang, Zhi

2017-09-19

Quantitative understanding the kinetics of toxic ion reactions with various heterogeneous ferrihydrite binding sites is crucial for accurately predicting the dynamic behavior of contaminants in environment. In this study, kinetics of As(V), Cr(VI), Cu(II), and Pb(II) adsorption and desorption on ferrihydrite was studied using a stirred-flow method, which showed that metal adsorption/desorption kinetics was highly dependent on the reaction conditions and varied significantly among four metals. High resolution scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed that all four metals were distributed within the ferrihydrite aggregates homogeneously after adsorption reactions. Based on the equilibrium model CD-MUSIC, we developed a novel unified kinetics model applicable for both cation and oxyanion adsorption and desorption on ferrihydrite, which is able to account for the heterogeneity of ferrihydrite binding sites, different binding properties of cations and oxyanions, and variations of solution chemistry. The model described the kinetic results well. We quantitatively elucidated how the equilibrium properties of the cation and oxyanion binding to various ferrihydrite sites and the formation of various surface complexes controlled the adsorption and desorption kinetics at different reaction conditions and time scales. Our study provided a unified modeling method for the kinetics of ion adsorption/desorption on ferrihydrite.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: equilibrium, kinetic, and crystallographic studies.

PubMed

Ledvina, P S; Tsai, A L; Wang, Z; Koehl, E; Quiocho, F A

1998-12-01

Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.

Characterization of [3H]LS-3-134, a Novel Arylamide Phenylpiperazine D3 Dopamine Receptor Selective Radioligand

PubMed Central

Rangel-Barajas, Claudia; Malik, Maninder; Taylor, Michelle; Neve, Kim A.; Mach, Robert H.; Luedtke, Robert R.

2014-01-01

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit a) high-affinity binding (Ki value 0.2 nM) at human D3 dopamine receptors, b) >100-fold D3 vs. D2 dopamine receptor subtype binding selectivity and c) low-affinity binding (Ki values >5,000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [3H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15 min at 37°C) and binds with high affinity to both human (Kd = 0.06 ± 0.01 nM) and rat (Kd = 0.2 ± 0.02 nM) D3 receptors expressed in HEK-293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [3H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies we propose that [3H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype. PMID:25041389

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

DNA-cisplatin binding mechanism peculiarities studied with single molecule stretching experiments

NASA Astrophysics Data System (ADS)

Crisafuli, F. A. P.; Cesconetto, E. C.; Ramos, E. B.; Rocha, M. S.

2012-02-01

We propose a method to determine the DNA-cisplatin binding mechanism peculiarities by monitoring the mechanical properties of these complexes. To accomplish this task, we have performed single molecule stretching experiments by using optical tweezers, from which the persistence and contour lengths of the complexes can be promptly measured. The persistence length of the complexes as a function of the drug total concentration in the sample was used to deduce the binding data, from which we show that cisplatin binds cooperatively to the DNA molecule, a point which so far has not been stressed in binding equilibrium studies of this ligand.

Time-resolved absorbance changes induced by fast acidification of bacteriorhodopsin in vesicle systems.

PubMed Central

Druckmann, S; Ottolenghi, M; Korenstein, R

1985-01-01

The direction of the accessibility to protons of the binding site in bacteriorhodopsin is of primary importance in elucidating the proton-pump mechanism. The problem is approached via the pH-dependent equilibrium bR560 in equilibrium bR605 in vesicles with preferentially oriented purple membranes. Fast acidification (stopped-flow) experiments with inside-out, monomeric, bR vesicles were carried out with and without a buffer enclosed in the vesicle interior. The results, showing a buffer-induced delay in the formation of bR605, indicate that the binding site is accessible to protons from the inside of the vesicles. We arrive at this conclusion also by working with inside-out trimeric vesicles in the presence and in the absence of H+ (and K+) ionophores. The results suggest that in Halobacterium halobium, the binding site and thus the retinal Schiff base are exposed to the outside of the cell. This conclusion is consistent with a pumping mechanism based on a light-induced pK change. PMID:3978185

Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate

PubMed Central

Warren, Graham B.; Tipton, Keith F.

1974-01-01

1. The reaction pathway for the decarboxylation of oxaloacetate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgADP− and Pi bind randomly, at equilibrium, followed by the binding of oxaloacetate. Pyruvate is released before the ordered steay-state release of HCO3− and MgATP2−. 3. These results are entirely consistent with studies on the carboxylation of pyruvate presented in the preceding paper (Warren & Tipton, 1974b) and together they allow a quantitative description of the reaction mechanism of pig liver pyruvate carboxylase. 4. In the absence of other substrates of the back reaction pig liver pyruvate carboxylase will decarboxylate oxaloacetate in a manner that is not inhibited by avidin. 5. Reciprocal plots involving oxaloacetate are non-linear curves, which suggest a negatively co-operative interaction between this substrate and the enzyme. PMID:4447613

Allostery and the dynamic oligomerization of porphobilinogen synthase

PubMed Central

Jaffe, Eileen K.; Lawrence, Sarah H.

2011-01-01

The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer* ⇔ 6mer*. The “*” represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH; the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer* relative to wild type. The 6mer* has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer*. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects. PMID:22037356

Tracking Equilibrium and Nonequilibrium Shifts in Data with TREND.

PubMed

Xu, Jia; Van Doren, Steven R

2017-01-24

Principal component analysis (PCA) discovers patterns in multivariate data that include spectra, microscopy, and other biophysical measurements. Direct application of PCA to crowded spectra, images, and movies (without selecting peaks or features) was shown recently to identify their equilibrium or temporal changes. To enable the community to utilize these capabilities with a wide range of measurements, we have developed multiplatform software named TREND to Track Equilibrium and Nonequilibrium population shifts among two-dimensional Data frames. TREND can also carry this out by independent component analysis. We highlight a few examples of finding concurrent processes. TREND extracts dual phases of binding to two sites directly from the NMR spectra of the titrations. In a cardiac movie from magnetic resonance imaging, TREND resolves principal components (PCs) representing breathing and the cardiac cycle. TREND can also reconstruct the series of measurements from selected PCs, as illustrated for a biphasic, NMR-detected titration and the cardiac MRI movie. Fidelity of reconstruction of series of NMR spectra or images requires more PCs than needed to plot the largest population shifts. TREND reads spectra from many spectroscopies in the most common formats (JCAMP-DX and NMR) and multiple movie formats. The TREND package thus provides convenient tools to resolve the processes recorded by diverse biophysical methods. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Plasmon-mediated binding forces on gold or silver homodimer and heterodimer

NASA Astrophysics Data System (ADS)

Liaw, Jiunn-Woei; Kuo, Ting-Yu; Kuo, Mao-Kuen

2016-02-01

This study theoretically investigates plasmon-mediated optical binding forces, which are exerted on metal homo or heterodimers, induced by the normal illumination of a linearly polarized plane wave or Gaussian beam. Using the multiple multipole method, we analyzed the optical force in terms of Maxwell's stress tensor for various interparticle distance at some specific wavelengths. Numerical results show that for a given wavelength there are several stable equilibrium distances between two nanoparticles (NPs) of a homodimer, which are slightly shorter than some integer multiples of the wavelength in medium, such that metal dimer acts as bonded together. At these specific interparticle distances, the optical force between dimer is null and serves a restoring force, which is repulsive and attractive, respectively, as the two NPs are moving closer to and away from each other. The spring constant of the restoring force at the first stable equilibrium is always the largest, indicating that the first stable equilibrium distance is the most stable one. Moreover, the central line (orientation) of a dimer tends to be perpendicular to the polarization of light. For the cases of heterodimers, the phenomenon of stable equilibrium interparticle distance still exists, except there is an extra net photophoretic force drifting the heterodimer as one. Moreover, gradient force provided by a Gaussian beam may reduce the stability of these equilibriums, so larger NPs are preferred to stabilize a dimer under illumination of Gaussian beam. The finding may pave the way for using optical manipulation on the gold or silver colloidal self-assembly.

I. Dissociation free energies of drug-receptor systems via non-equilibrium alchemical simulations: a theoretical framework.

PubMed

Procacci, Piero

2016-06-01

In this contribution I critically revise the alchemical reversible approach in the context of the statistical mechanics theory of non-covalent bonding in drug-receptor systems. I show that most of the pitfalls and entanglements for the binding free energy evaluation in computer simulations are rooted in the equilibrium assumption that is implicit in the reversible method. These critical issues can be resolved by using a non-equilibrium variant of the alchemical method in molecular dynamics simulations, relying on the production of many independent trajectories with a continuous dynamical evolution of an externally driven alchemical coordinate, completing the decoupling of the ligand in a matter of a few tens of picoseconds rather than nanoseconds. The absolute binding free energy can be recovered from the annihilation work distributions by applying an unbiased unidirectional free energy estimate, on the assumption that any observed work distribution is given by a mixture of normal distributions, whose components are identical in either direction of the non-equilibrium process, with weights regulated by the Crooks theorem. I finally show that the inherent reliability and accuracy of the unidirectional estimate of the decoupling free energies, based on the production of a few hundreds of non-equilibrium independent sub-nanosecond unrestrained alchemical annihilation processes, is a direct consequence of the funnel-like shape of the free energy surface in molecular recognition. An application of the technique to a real drug-receptor system is presented in the companion paper.

The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry

PubMed Central

Sun, Lijun; Gidley, Michael J.

2017-01-01

Scope This study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA). Methods and results The interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K ic), quenching constant (K FQ) and binding constant (K itc). Conclusion The combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA. PMID:28618113

Conformational Contribution to Thermodynamics of Binding in Protein-Peptide Complexes through Microscopic Simulation

PubMed Central

Das, Amit; Chakrabarti, J.; Ghosh, Mahua

2013-01-01

We extract the thermodynamics of conformational changes in biomacromolecular complexes from the distributions of the dihedral angles of the macromolecules. These distributions are obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. The conformational thermodynamics data we obtained for calmodulin-peptide complexes using our methodology corroborate well with the experimentally observed conformational and binding entropies. The conformational free-energy changes and their contributions for different peptide-binding regions of calmodulin are evaluated microscopically. PMID:23528087

An Undergraduate Laboratory Experiment that Utilizes a Glass Fiber Filter Assay to Determine the Steroid Specificity and Equilibrium Binding Properties of Glucocorticoid Receptors.

ERIC Educational Resources Information Center

John, Nancy J.; Firestone, Gary L.

1987-01-01

Describes two complementary laboratory exercises that use the glass fiber assay to assess receptor specificity and hormone binding affinity in rat liver cytoplasmic extracts. Details the methods, materials and protocol of the experiments. Discusses the basic concepts illustrated and the feasibility of using the experiments at the undergraduate…

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

PubMed Central

Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

2014-01-01

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

A model for the salt effect on adsorption equilibrium of basic protein to dye-ligand affinity adsorbent.

PubMed

Zhang, Songping; Sun, Yan

2004-01-01

A model describing the salt effect on adsorption equilibrium of a basic protein, lysozyme, to Cibacron Blue 3GA-modified Sepharose CL-6B (CB-Sepharose) has been developed. In this model, it is assumed that the presence of salt causes a fraction of dye-ligand molecules to lodge to the surface of the agarose gel, resulting from the induced strong hydrophobic interaction between dye ligand and agarose matrix. The salt effect on the lodging of dye-ligand is expressed by the equilibrium between salt and dye-ligand. For the interactions between protein and vacant binding sites, stoichiometric equations based either on cation exchanges or on hydrophobic interactions are proposed since the CB dye can be regarded as a cation exchanger contributed by the sulfonate groups on it. Combining with the basic concept of steric mass-action theory for ion exchange, which considers both the multipoint nature and the macromolecular steric shielding of protein adsorption, an explicit isotherm for protein adsorption equilibrium on the dye-ligand adsorbent is formulated, involving salt concentration as a variable. Analysis of the model parameters has yielded better understanding of the mechanism of salt effects on adsorption of the basic protein. Moreover, the model predictions are in good agreement with the experimental data over a wide range of salt and ligand concentrations, indicating the predictive nature of the model.

Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement.

PubMed

Deng, Jianjun; Chen, Fei; Fan, Daidi; Zhu, Chenhui; Ma, Xiaoxuan; Xue, Wenjiao

2013-10-01

Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein-iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (nb) and apparent association constant (Kapp) between iron and phosphorylated HLC were measured at nb=23.7 and log Kapp=4.57, respectively. The amount of iron (Fe(2+) sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates. © 2013.

A facilitated diffusion model constrained by the probability isotherm: a pedagogical exercise in intuitive non-equilibrium thermodynamics.

PubMed

Chapman, Brian

2017-06-01

This paper seeks to develop a more thermodynamically sound pedagogy for students of biological transport than is currently available from either of the competing schools of linear non-equilibrium thermodynamics (LNET) or Michaelis-Menten kinetics (MMK). To this end, a minimal model of facilitated diffusion was constructed comprising four reversible steps: cis- substrate binding, cis → trans bound enzyme shuttling, trans -substrate dissociation and trans → cis free enzyme shuttling. All model parameters were subject to the second law constraint of the probability isotherm, which determined the unidirectional and net rates for each step and for the overall reaction through the law of mass action. Rapid equilibration scenarios require sensitive 'tuning' of the thermodynamic binding parameters to the equilibrium substrate concentration. All non-equilibrium scenarios show sigmoidal force-flux relations, with only a minority of cases having their quasi -linear portions close to equilibrium. Few cases fulfil the expectations of MMK relating reaction rates to enzyme saturation. This new approach illuminates and extends the concept of rate-limiting steps by focusing on the free energy dissipation associated with each reaction step and thereby deducing its respective relative chemical impedance. The crucial importance of an enzyme's being thermodynamically 'tuned' to its particular task, dependent on the cis- and trans- substrate concentrations with which it deals, is consistent with the occurrence of numerous isoforms for enzymes that transport a given substrate in physiologically different circumstances. This approach to kinetic modelling, being aligned with neither MMK nor LNET, is best described as intuitive non-equilibrium thermodynamics, and is recommended as a useful adjunct to the design and interpretation of experiments in biotransport.

A facilitated diffusion model constrained by the probability isotherm: a pedagogical exercise in intuitive non-equilibrium thermodynamics

PubMed Central

2017-01-01

This paper seeks to develop a more thermodynamically sound pedagogy for students of biological transport than is currently available from either of the competing schools of linear non-equilibrium thermodynamics (LNET) or Michaelis–Menten kinetics (MMK). To this end, a minimal model of facilitated diffusion was constructed comprising four reversible steps: cis-substrate binding, cis→trans bound enzyme shuttling, trans-substrate dissociation and trans→cis free enzyme shuttling. All model parameters were subject to the second law constraint of the probability isotherm, which determined the unidirectional and net rates for each step and for the overall reaction through the law of mass action. Rapid equilibration scenarios require sensitive ‘tuning’ of the thermodynamic binding parameters to the equilibrium substrate concentration. All non-equilibrium scenarios show sigmoidal force–flux relations, with only a minority of cases having their quasi-linear portions close to equilibrium. Few cases fulfil the expectations of MMK relating reaction rates to enzyme saturation. This new approach illuminates and extends the concept of rate-limiting steps by focusing on the free energy dissipation associated with each reaction step and thereby deducing its respective relative chemical impedance. The crucial importance of an enzyme's being thermodynamically ‘tuned’ to its particular task, dependent on the cis- and trans-substrate concentrations with which it deals, is consistent with the occurrence of numerous isoforms for enzymes that transport a given substrate in physiologically different circumstances. This approach to kinetic modelling, being aligned with neither MMK nor LNET, is best described as intuitive non-equilibrium thermodynamics, and is recommended as a useful adjunct to the design and interpretation of experiments in biotransport. PMID:28680687

Mapping heat exchange in an allosteric protein.

PubMed

Gupta, Shaweta; Auerbach, Anthony

2011-02-16

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

Curcumin Binding to Beta Amyloid: A Computational Study.

PubMed

Rao, Praveen P N; Mohamed, Tarek; Teckwani, Karan; Tin, Gary

2015-10-01

Curcumin, a chemical constituent present in the spice turmeric, is known to prevent the aggregation of amyloid peptide implicated in the pathophysiology of Alzheimer's disease. While curcumin is known to bind directly to various amyloid aggregates, no systematic investigations have been carried out to understand its ability to bind to the amyloid aggregates including oligomers and fibrils. In this study, we constructed computational models of (i) Aβ hexapeptide (16) KLVFFA(21) octamer steric-zipper β-sheet assembly and (ii) full-length Aβ fibril β-sheet assembly. Curcumin binding in these models was evaluated by molecular docking and molecular dynamics (MD) simulation studies. In both the models, curcumin was oriented in a linear extended conformation parallel to fiber axis and exhibited better stability in the Aβ hexapeptide (16) KLVFFA(21) octamer steric-zipper model (Ebinding  = -10.05 kcal/mol) compared to full-length Aβ fibril model (Ebinding  = -3.47 kcal/mol). Analysis of MD trajectories of curcumin bound to full-length Aβ fibril shows good stability with minimum Cα-atom RMSD shifts. Interestingly, curcumin binding led to marked fluctuations in the (14) HQKLVFFA(21) region that constitute the fibril spine with RMSF values ranging from 1.4 to 3.6 Å. These results show that curcumin binding to Aβ shifts the equilibrium in the aggregation pathway by promoting the formation of non-toxic aggregates. © 2015 John Wiley & Sons A/S.

Direct Binding of the Corrector VX-809 to Human CFTR NBD1: Evidence of an Allosteric Coupling between the Binding Site and the NBD1:CL4 Interface.

PubMed

Hudson, Rhea P; Dawson, Jennifer E; Chong, P Andrew; Yang, Zhengrong; Millen, Linda; Thomas, Philip J; Brouillette, Christie G; Forman-Kay, Julie D

2017-08-01

Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of β -strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (T m ), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative T m shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR. Copyright © 2017 by The Author(s).

Intrinsically Disordered Energy Landscapes

PubMed Central

Chebaro, Yassmine; Ballard, Andrew J.; Chakraborty, Debayan; Wales, David J.

2015-01-01

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such ‘intrinsically disordered’ landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an -helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium. PMID:25999294

Kinetic contribution to extracellular Na+/K+ selectivity in the Na+/K+ pump.

PubMed

Vleeskens, Elizabeth; Clarke, Ronald J

2018-05-01

The sodium potassium pump (Na + ,K + -ATPase) shows a high selectivity for K + over Na + binding from the extracellular medium. To understand the K + selectivity in the presence of a high concentration of competing Na + ions requires consideration of more than just ion binding affinities. Here, equilibrium-based calculations of the extracellular occupation of the Na + ,K + -ATPase transport sites by Na + and K + are compared to fluxes through Na + and K + transport pathways. The results show that, under physiological conditions, there is a 332-fold selectivity for pumping of K + from the extracellular medium into the cytoplasm relative to Na + , whereas equilibrium calculations alone predict only a 7.5-fold selectivity for K + . Thus, kinetic effects make a major contribution to the determination of extracellular K + selectivity.

Effect of antemortem and postmortem factors on ( sup 3 H)MK-801 binding in the human brain: Transient elevation during early childhood

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kornhuber, J.; Mack-Burkhardt, F.; Konradi, C.

1989-01-01

The effect of a number of antemortem and postmortem factors on ({sup 3}H)MK-801 binding was investigated under equilibrium conditions in the frontal cortex of human brains of 38 controls. Binding values transiently increased during the early postnatal period reaching a maximum at the age of about 2 years. After age 10 years ({sup 3}H)MK-801 binding sites disappeared at 5.7% per decade. The storage time of brain tissue had a reducing effect on these binding sites. There was no effect of gender, brain weight or postmortem time interval and the binding sites were bilaterally symmetrically distributed in the frontal cortex.

Thermodynamics of Alkanethiol Self-Assembled Monolayer Assembly on Pd Surfaces.

PubMed

Kumar, Gaurav; Van Cleve, Timothy; Park, Jiyun; van Duin, Adri; Medlin, J Will; Janik, Michael J

2018-06-05

We investigate the structure and binding energy of alkanethiolate self-assembled monolayers (SAMs) on Pd (111), Pd (100), and Pd (110) facets at different coverages. Dispersion-corrected density functional theory calculations are used to correlate the binding energy of alkanethiolates with alkyl chain length and coverage. The equilibrium coverage of thiolate layers strongly prefers 1/3 monolayer (ML) on the Pd (111) surface. The coverage of thiolates varies with chemical potential on Pd (100) and Pd (110), increasing from 1/3 to 1/2 ML on (100) and from 1/4 to 1/2 ML on (110) as the thiol chemical potential is increased. Higher coverages are driven by attractive dispersion interactions between the extended alkyl chains, such that transitions to higher coverages occur at lower thiol chemical potentials for longer chain thiolates. Stronger adsorption to the Pd (100) surface causes the equilibrium Wulff construction of Pd particles to take on a cubic shape upon saturation with thiols. The binding of H, O, and CO adsorbates is weakened as the thiolate coverage is increased, with saturation coverages causing unfavorable binding of O and CO on Pd (100) and weakened binding on other facets. Temperature-dependent CO diffuse reflectance infrared Fourier transform spectroscopy experiments are used to corroborate the weakened binding of CO in the presence of thiolate SAMs of varying surface density. Preliminary results of multiscale modeling efforts on the Pd-thiol system using a reactive force field, ReaxFF, are also discussed.

The Conformational Variability of FimH: Which Conformation Represents the Therapeutic Target?

PubMed

Eris, Deniz; Preston, Roland C; Scharenberg, Meike; Hulliger, Fabian; Abgottspon, Daniela; Pang, Lijuan; Jiang, Xiaohua; Schwardt, Oliver; Ernst, Beat

2016-06-02

FimH is a bacterial lectin found at the tips of type 1 pili of uropathogenic Escherichia coli (UPEC). It mediates shear-enhanced adhesion to mannosylated surfaces. Binding of UPEC to urothelial cells initiates the infection cycle leading to urinary tract infections (UTIs). Antiadhesive glycomimetics based on α-d-mannopyranose offer an attractive alternative to the conventional antibiotic treatment because they do not induce a selection pressure and are therefore expected to have a reduced resistance potential. Genetic variation of the fimH gene in clinically isolated UPEC has been associated with distinct mannose binding phenotypes. For this reason, we investigated the mannose binding characteristics of four FimH variants with mannose-based ligands under static and hydrodynamic conditions. The selected FimH variants showed individually different binding behavior under both sets of conditions as a result of the conformational variability of FimH. Clinically relevant FimH variants typically exist in a dynamic conformational equilibrium. Additionally, we evaluated inhibitory potencies of four FimH antagonists representing different structural classes. Inhibitory potencies of three of the tested antagonists were dependent on the binding phenotype and hence on the conformational equilibrium of the FimH variant. However, the squarate derivative was the notable exception and inhibited FimH variants irrespective of their binding phenotype. Information on antagonist affinities towards various FimH variants has remained largely unconsidered despite being essential for successful antiadhesion therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PubMed

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Theoretical study of the coordination behavior of formate and formamidoximate with dioxovanadium( v ) cation: implications for selectivity towards uranyl

DOE PAGES

Mehio, Nada; Johnson, J. Casey; Dai, Sheng; ...

2015-10-28

Poly(acrylamidoxime)-based fibers bearing random mixtures of carboxylate and amidoxime groups are the most widely utilized materials for extracting uranium from seawater. However, the competition between uranyl (UO 2 2+) and vanadium ions poses a significant challenge to the industrial mining of uranium from seawater using the current generation of adsorbents. To design more selective adsorbents, a detailed understanding of how major competing ions interact with carboxylate and amidoxime ligands is required. In this work, we employ density functional theory (DFT) and wave-function methods to investigate potential binding motifs of the dioxovanadium ion, VO 2 +, with water, formate, and formamidoximatemore » ligands. Employing higher level of theory calculations (CCSD(T)) resolve the existing controversy between the experimental results and previous DFT calculations for the structure of the hydrated VO 2 + ion. Consistent with the EXAFS data, CCSD(T) calculations predict higher stability of the distorted octahedral geometry of VO 2 +(H 2O) 4 compared to the five-coordinate complex with a single water molecule in the second hydration shell, while all seven tested DFT methods yield the reverse stability of the two conformations. Analysis of the relative stabilities of formate-VO 2 + complexes indicates that both monodentate and bidentate forms may coexist in thermodynamic equilibrium in solution, with the equilibrium balance leaning more towards the formation of monodentate species. Investigations of VO 2 + coordination with the formamidoximate anion has revealed the existence of seven possible binding motifs, four of which are within ~ 4.0 kcal/mol of each other. Calculations establish that the most stable binding motif entails the coordination of oxime oxygen and amide nitrogen atoms via a tautomeric rearrangement of amidoxime to imino hydroxylamine. Lastly, the difference in the most stable VO 2 + and UO 2 2+ binding conformation has important implications for the design of more selective UO 2 2+ ligands.« less

Binding of corroded ions to human saliva.

PubMed

Mueller, H J

1985-05-01

Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

Timescale analysis of rule-based biochemical reaction networks

PubMed Central

Klinke, David J.; Finley, Stacey D.

2012-01-01

The flow of information within a cell is governed by a series of protein-protein interactions that can be described as a reaction network. Mathematical models of biochemical reaction networks can be constructed by repetitively applying specific rules that define how reactants interact and what new species are formed upon reaction. To aid in understanding the underlying biochemistry, timescale analysis is one method developed to prune the size of the reaction network. In this work, we extend the methods associated with timescale analysis to reaction rules instead of the species contained within the network. To illustrate this approach, we applied timescale analysis to a simple receptor-ligand binding model and a rule-based model of Interleukin-12 (IL-12) signaling in näive CD4+ T cells. The IL-12 signaling pathway includes multiple protein-protein interactions that collectively transmit information; however, the level of mechanistic detail sufficient to capture the observed dynamics has not been justified based upon the available data. The analysis correctly predicted that reactions associated with JAK2 and TYK2 binding to their corresponding receptor exist at a pseudo-equilibrium. In contrast, reactions associated with ligand binding and receptor turnover regulate cellular response to IL-12. An empirical Bayesian approach was used to estimate the uncertainty in the timescales. This approach complements existing rank- and flux-based methods that can be used to interrogate complex reaction networks. Ultimately, timescale analysis of rule-based models is a computational tool that can be used to reveal the biochemical steps that regulate signaling dynamics. PMID:21954150

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter.

PubMed

Rosenberry, Terrone L; Sonoda, Leilani K; Dekat, Sarah E; Cusack, Bernadette; Johnson, Joseph L

2008-12-09

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

Analysis of the reaction of carbachol with acetylcholinesterase with thioflavin T as a coupled fluorescence reporter†

PubMed Central

Rosenberry, Terrone L.; Sonoda, Leilani K.; Dekat, Sarah E.; Cusack, Bernadette; Johnson, Joseph L.

2009-01-01

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC) which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analog of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging, because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site. PMID:19006330

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

A model of lipid-free Apolipoprotein A-I revealed by iterative molecular dynamics simulation

DOE PAGES

Zhang, Xing; Lei, Dongsheng; Zhang, Lei; ...

2015-03-20

Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore,more » by integrating various experimental results, we proposed a new structural model for lipidfree apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.« less

Contrasting guest binding interaction of cucurbit[7-8]urils with neutral red dye: controlled exchange of multiple guests.

PubMed

Shaikh, Mhejabeen; Choudhury, Sharmistha Dutta; Mohanty, Jyotirmayee; Bhasikuttan, Achikanath C; Pal, Haridas

2010-07-14

Interactions among macrocyclic hosts and dyes/drugs have been explored extensively for their direct usage in controlled uptake and release of large number of potential drug molecules. In this paper we report the non-covalent interaction of cucurbit[8]uril macrocycle (CB8) with a biologically important dye, neutral red, by absorption and fluorescence spectroscopy. A comparative analysis with the complexation behaviour of the dye with CB7, the lower homologue of CB8, indicates contrasting guest binding behaviour with significant changes in the photophysical characteristics of the dye. While CB7 interaction leads to a 1 ratio 1 stoichiometry resulting in approximately 6 fold enhancement in the fluorescence emission of the dye, CB8 displays signatures for a 1 ratio 2 host-guest stoichiometry with drastic reduction in the fluorescence emission. Apart from the evaluation of approximately 2 unit shift in the protolytic equilibrium on complexation (pK(a) shift), the measurements with tryptophan established a selective guest exchange to favour a co-localized dimer inside the CB8 cavity. In a protein medium (BSA), the 1 ratio 2 complex was converted to a 1 ratio 1 ratio 1 CB8-NRH(+)-BSA complex. The finding that NRH(+) can be transferred from CB8 to BSA, even though the binding constant for NRH(+)-CB8 is much higher than NRH(+)-BSA, is projected for a controlled slow release of NRH(+) towards BSA. Since the release and activity of drugs can be controlled by regulating the protolytic equilibrium, the macromolecular encapsulation and release of NRH(+) demonstrated here provide information relevant to host-guest based drug delivery systems and its applications.

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.

PubMed

Thomas, Sabrina; Bakkes, Patrick J; Smits, Sander H J; Schmitt, Lutz

2014-09-01

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca(2+) binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6-7M urea, relates to Ca(2+) dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

Temperature-Dependent Estimation of Gibbs Energies Using an Updated Group-Contribution Method.

PubMed

Du, Bin; Zhang, Zhen; Grubner, Sharon; Yurkovich, James T; Palsson, Bernhard O; Zielinski, Daniel C

2018-06-05

Reaction-equilibrium constants determine the metabolite concentrations necessary to drive flux through metabolic pathways. Group-contribution methods offer a way to estimate reaction-equilibrium constants at wide coverage across the metabolic network. Here, we present an updated group-contribution method with 1) additional curated thermodynamic data used in fitting and 2) capabilities to calculate equilibrium constants as a function of temperature. We first collected and curated aqueous thermodynamic data, including reaction-equilibrium constants, enthalpies of reaction, Gibbs free energies of formation, enthalpies of formation, entropy changes of formation of compounds, and proton- and metal-ion-binding constants. Next, we formulated the calculation of equilibrium constants as a function of temperature and calculated the standard entropy change of formation (Δ f S ∘ ) using a model based on molecular properties. The median absolute error in estimating Δ f S ∘ was 0.013 kJ/K/mol. We also estimated magnesium binding constants for 618 compounds using a linear regression model validated against measured data. We demonstrate the improved performance of the current method (8.17 kJ/mol in median absolute residual) over the current state-of-the-art method (11.47 kJ/mol) in estimating the 185 new reactions added in this work. The efforts here fill in gaps for thermodynamic calculations under various conditions, specifically different temperatures and metal-ion concentrations. These, to our knowledge, new capabilities empower the study of thermodynamic driving forces underlying the metabolic function of organisms living under diverse conditions. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The use of analytical sedimentation velocity to extract thermodynamic linkage.

PubMed

Cole, James L; Correia, John J; Stafford, Walter F

2011-11-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. Copyright © 2011 Elsevier B.V. All rights reserved.

The use of analytical sedimentation velocity to extract thermodynamic linkage

PubMed Central

Cole, James L.; Correia, John J.; Stafford, Walter F.

2011-01-01

For 25 years, the Gibbs Conference on Biothermodynamics has focused on the use of thermodynamics to extract information about the mechanism and regulation of biological processes. This includes the determination of equilibrium constants for macromolecular interactions by high precision physical measurements. These approaches further reveal thermodynamic linkages to ligand binding events. Analytical ultracentrifugation has been a fundamental technique in the determination of macromolecular reaction stoichiometry and energetics for 85 years. This approach is highly amenable to the extraction of thermodynamic couplings to small molecule binding in the overall reaction pathway. In the 1980’s this approach was extended to the use of sedimentation velocity techniques, primarily by the analysis of tubulin-drug interactions by Na and Timasheff. This transport method necessarily incorporates the complexity of both hydrodynamic and thermodynamic nonideality. The advent of modern computational methods in the last 20 years has subsequently made the analysis of sedimentation velocity data for interacting systems more robust and rigorous. Here we review three examples where sedimentation velocity has been useful at extracting thermodynamic information about reaction stoichiometry and energetics. Approaches to extract linkage to small molecule binding and the influence of hydrodynamic nonideality are emphasized. These methods are shown to also apply to the collection of fluorescence data with the new Aviv FDS. PMID:21703752

Calculating binding free energies for protein-carbohydrate complexes.

PubMed

Hadden, Jodi A; Tessier, Matthew B; Fadda, Elisa; Woods, Robert J

2015-01-01

A variety of computational techniques may be applied to compute theoretical binding free energies for protein-carbohydrate complexes. Elucidation of the intermolecular interactions, as well as the thermodynamic effects, that contribute to the relative strength of receptor binding can shed light on biomolecular recognition, and the resulting initiation or inhibition of a biological process. Three types of free energy methods are discussed here, including MM-PB/GBSA, thermodynamic integration, and a non-equilibrium alternative utilizing SMD. Throughout this chapter, the well-known concanavalin A lectin is employed as a model system to demonstrate the application of these methods to the special case of carbohydrate binding.

Complexation of iron hexacyanides by cytochrome c. Evidence for electron exchange at the exposed heme edge.

PubMed

Stellwagen, E; Cass, R D

1975-03-25

Electrostatic binding of at least two anionic iron hexacyanides to cationic horse heart cytochrome c was demonstrated by equilibrium dialysis measurements. No binding was detected following trifluoroacetylation of all of the 19 lysine residues. Replacement of the natural heme iron ligand methionine 80 by the alternative intrinsic ligand lysine 79 but not the extrinsic ligand imidazole resulted in the loss of one hexacyanide binding site. It is proposed that this site is located at the exposed heme edge and is functional in electron exchange.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Surface acoustic wave oxygen sensor

NASA Technical Reports Server (NTRS)

Collman, James P.; Oglesby, Donald M.; Upchurch, Billy T.; Leighty, Bradley D.; Zhang, Xumu; Herrmann, Paul C.

1994-01-01

A surface acoustic wave (SAW) device that responds to oxygen pressure was developed by coating a 158 MHz quartz surface acoustic wave (SAW) device with an oxygen binding agent. Two types of coatings were used. One type was prepared by dissolving an oxygen binding agent in a toluene solution of a copolymer containing the axial ligand. A second type was prepared with an oxygen binding porphyrin solution containing excess axial ligand without a polymer matrix. In the polymer based coatings, the copolymer served to provide the axial ligand to the oxygen binding agent and as a coating matrix on the surface of the SAW device. The oxygen sensing SAW device has been shown to bind oxygen following a Langmuir isotherm and may be used to measure the equilibrium constant of the oxygen binding compound in the coating matrix.

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum.

PubMed

Stratton, Christopher F; Namanja-Magliano, Hilda A; Cameron, Scott A; Schramm, Vern L

2015-10-16

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

PubMed

Maung-Maung-Thwin; Gopalakrishnakone, P; Yuen, R; Tan, C H

1996-02-01

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

PubMed

Nobrega, R Paul; Brown, Michael; Williams, Cody; Sumner, Chris; Estep, Patricia; Caffry, Isabelle; Yu, Yao; Lynaugh, Heather; Burnina, Irina; Lilov, Asparouh; Desroches, Jordan; Bukowski, John; Sun, Tingwan; Belk, Jonathan P; Johnson, Kirt; Xu, Yingda

2017-10-01

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

Exploring Strategies for Labeling Viruses with Gold Nanoclusters through Non-equilibrium Molecular Dynamics Simulations.

PubMed

Pohjolainen, Emmi; Malola, Sami; Groenhof, Gerrit; Häkkinen, Hannu

2017-09-20

Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au 102 pMBA 44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.

A distal point mutation in the streptavidin-biotin complex preserves structure but diminishes binding affinity: experimental evidence of electronic polarization effects?

PubMed

Baugh, Loren; Le Trong, Isolde; Cerutti, David S; Gülich, Susanne; Stayton, Patrick S; Stenkamp, Ronald E; Lybrand, Terry P

2010-06-08

We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule; however, all neighboring side chain positions are preserved, and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal a reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen bonding network in the biotin binding pocket.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting results of other DNA sequences (Table 2S); calculation of dissociation constants (Fig. 3S, 4S; Table 2S); geometric model for quantitation of DNA bending angle induced by specific IHF binding (Fig. 4S); customized flow cell assembly (Fig. 5S); real-time measurement of average fluorophore height change by SSFM (Fig. 6S); summary of binding parameters obtained from additive isotherm model fitting (Table 3S); average surface densities of 10 dsDNA spots and bound IHF at equilibrium (Table 4S); effects of surface densities on the binding and bending of dsDNA (Tables 5S, 6S and Fig. 7S-10S). See DOI: 10.1039/c5nr06785e

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

PubMed

Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

2017-06-23

The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

Local nature of impurity induced spin-orbit torques

NASA Astrophysics Data System (ADS)

Nikolaev, Sergey; Kalitsov, Alan; Chshiev, Mairbec; Mryasov, Oleg

Spin-orbit torques are of a great interest due to their potential applications for spin electronics. Generally, it originates from strong spin orbit coupling of heavy 4d/5d elements and its mechanism is usually attributed either to the Spin Hall effect or Rashba spin-orbit coupling. We have developed a quantum-mechanical approach based on the non-equilibrium Green's function formalism and tight binding Hamiltonian model to study spin-orbit torques and extended our theory for the case of extrinsic spin-orbit coupling induced by impurities. For the sake of simplicity, we consider a magnetic material on a two dimensional lattice with a single non-magnetic impurity. However, our model can be easily extended for three dimensional layered heterostructures. Based on our calculations, we present the detailed analysis of the origin of local spin-orbit torques and persistent charge currents around the impurity, that give rise to spin-orbit torques even in equilibrium and explain the existence of anisotropy.

Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, K.A.; LaBarbera, A.R.

1988-11-01

The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase inmore » receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.« less

Selective Solid-Phase Extraction of Zinc(II) from Environmental Water Samples Using Ion Imprinted Activated Carbon.

PubMed

Moniri, Elham; Panahi, Homayon Ahmad; Aghdam, Khaledeh; Sharif, Amir Abdollah Mehrdad

2015-01-01

A simple ion imprinted amino-functionalized sorbent was synthesized by coupling activated carbon with iminodiacetic acid, a functional compound for metal chelating, through cyanoric chloride spacer. The resulting sorbent has been characterized using FTIR spectroscopy, elemental analysis, and thermogravimetric analysis and evaluated for the preconcentration and determination of trace Zn(II) in environmental water samples. The optimum pH value for sorption of the metal ion was 6-7.5. The sorption capacity of the functionalized sorbent was 66.6 mg/g. The chelating sorbent can be reused for 10 cycles of sorption-desorption without any significant change in sorption capacity. A recovery of 100% was obtained for the metal ion with 0.5 M nitric acid as the eluent. Compared with nonimprinted polymer particles, the prepared Zn-imprinted sorbent showed high adsorption capacity, significant selectivity, and good site accessibility for Zn(II). Scatchard analysis revealed that the homogeneous binding sites were formed in the polymer. The equilibrium sorption data of Zn(II) by modified resin were analyzed by Langmuir, Freundlich, Temkin, and Redlich-Peterson models. Based on equilibrium adsorption data, the Langmuir, Freundlich, and Temkin constants were determined as 0.139, 12.82, and 2.34, respectively, at 25°C.

Comparative evaluation of in vitro efficacy of colesevelam hydrochloride tablets.

PubMed

Krishnaiah, Yellela S R; Yang, Yongsheng; Bykadi, Srikant; Sayeed, Vilayat A; Khan, Mansoor A

2014-09-01

Colesevelam hydrochloride is used as an adjunct to diet and exercise to reduce elevated low-density lipoprotein (LDL) cholesterol in patients with primary hyperlipidemia as well as to improve glycemic control in patients with type 2 diabetes. This is likely to result in submission of abbreviated new drug applications (ANDA). This study was conducted to compare the efficacy of two tablet products of colesevelam hydrochloride based on the in vitro binding of bile acid sodium salts of glycocholic acid (GC), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA). Kinetic binding study was carried out with constant initial bile salt concentrations as a function of time. Equilibrium binding studies were conducted under conditions of constant incubation time and varying initial concentrations of bile acid sodium salts. The unbound concentration of bile salts was determined in the samples of these studies. Langmuir equation was utilized to calculate the binding constants k1 and k2. The amount of the three bile salts bound to both the products reached equilibrium at 3 h. The similarity factor (f2) was 99.5 based on the binding profile of total bile salts to the test and reference colesevelam tablets as a function of time. The 90% confidence interval for the test to reference ratio of k2 values were 96.06-112.07 which is within the acceptance criteria of 80-120%. It is concluded from the results that the test and reference tablets of colesevelam hydrochloride showed a similar in vitro binding profile and capacity to bile salts.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

PubMed Central

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

PubMed

Xie, Y; Cohen, J B

2001-01-26

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

Structural Basis for Activation of Fatty Acid-binding Protein 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillilan,R.; Ayers, S.; Noy, N.

2007-01-01

Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPAR{gamma} in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicatesmore » that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.« less

Binding assessment of two arachidonic-based synthetic derivatives of adrenalin with β-lactoglobulin: Molecular modeling and chemometrics approach.

PubMed

Gholami, S; Bordbar, A K; Akvan, N; Parastar, H; Fani, N; Gretskaya, N M; Bezuglov, V V; Haertlé, T

2015-12-01

A computational approach to predict the main binding modes of two adrenalin derivatives, arachidonoyl adrenalin (AA-AD) and arachidonoyl noradrenalin (AA-NOR) with the β-lactoglubuline (BLG) as a nano-milk protein carrier is presented and assessed by comparison to the UV-Vis absorption spectroscopic data using chemometric analysis. Analysis of the spectral data matrices by using the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm led to the pure concentration calculation and spectral profiles resolution of the chemical constituents and the apparent equilibrium constants computation. The negative values of entropy and enthalpy changes for both compound indicated the essential role of hydrogen bonding and van der Waals interactions as main driving forces in stabilizing protein-ligand complex. Computational studies predicted that both derivatives are situated in the calyx pose and remained in that pose during the whole time of simulation with no any significant protein structural changes which pointed that the BLG could be considered as a suitable carrier for these catecholamine compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Studies on molecular structure, vibrational spectra and molecular docking analysis of 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate

NASA Astrophysics Data System (ADS)

Suresh, D. M.; Amalanathan, M.; Hubert Joe, I.; Bena Jothy, V.; Diao, Yun-Peng

2014-09-01

The molecular structure, vibrational analysis and molecular docking analysis of the 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate (MDDNAB) molecule have been carried out using FT-IR and FT-Raman spectroscopic techniques and DFT method. The equilibrium geometry, harmonic vibrational wave numbers, various bonding features have been computed using density functional method. The calculated molecular geometry has been compared with experimental data. The detailed interpretation of the vibrational spectra has been carried out by using VEDA program. The hyper-conjugative interactions and charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The simulated FT-IR and FT-Raman spectra satisfactorily coincide with the experimental spectra. The PES and charge analysis have been made. The molecular docking was done to identify the binding energy and the Hydrogen bonding with the cancer protein molecule.

Specific binding of [(18)F]fluoroethyl-harmol to monoamine oxidase A in rat brain cryostat sections, and compartmental analysis of binding in living brain.

PubMed

Maschauer, Simone; Haller, Adelina; Riss, Patrick J; Kuwert, Torsten; Prante, Olaf; Cumming, Paul

2015-12-01

We investigated [(18)F]fluoroethyl-harmol ([(18)F]FEH) as a reversible and selective ligand for positron emission tomography (PET) studies of monoamine oxidase A (MAO-A). Binding of [(18)F]FEH in rat brain cryostat sections indicated high affinity (KD = 3 nM), and density (Bmax; 600 pmol/g). The plasma free fraction was 45%, and untransformed parent constituted only 13% of plasma radioactivity at 10 min after injection. Compartmental analysis of PET recordings in pargyline-treated rats showed high permeability to brain (K1; 0.32 mL/g/min) and slow washout (k2; 0.024/min), resulting in a uniformly high equilibrium distribution volume (VD; 20 mL/g). Using this VD to estimate unbound ligand in brain of untreated rats, the binding potential ranged from 4.2 in cerebellum to 7.2 in thalamus. We also calculated maps of rats receiving [(18)F]FEH at a range of specific activities, and then estimated saturation binding parameters in the living brain. In thalamus, striatum and frontal cortex KD was globally close to 300 nM and Bmax was close to 1600 pmol/g; the 100-fold discrepancy in affinity suggests a very low free fraction for [(18)F]FEH in the living brain. Based on a synthesis of findings, we calculate the endogenous dopamine concentration to be 0.4 μM in the striatal compartment containing MAO-A, thus unlikely to exert competition against [(18)F]FEH binding in vivo. In summary, [(18)F]FEH has good properties for the detection of MAO-A in the rat brain by PET, and may present logistic advantages for clinical research at centers lacking a medical cyclotron. We made a compartmental analysis of [(18)F]fluoroethylharmol ([(18)F]FEH) binding to monoamine oxidase A (MAO-A) in living rat brain and estimated the saturation binding parameters from the binding potential (BPND). The Bmax was of comparable magnitude to that in vitro, but with apparent affinity (300 nM), it was 100-fold lower in vivo. PET imaging with [(18) F]FEH is well suited for quantitation of MAO-A in living brain. © 2015 International Society for Neurochemistry.

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase.

PubMed Central

Babes, A; Fendler, K

2000-01-01

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P. PMID:11053130

Equilibrium isotope effects on noncovalent interactions in a supramolecular host-guest system.

PubMed

Mugridge, Jeffrey S; Bergman, Robert G; Raymond, Kenneth N

2012-02-01

The self-assembled supramolecular complex [Ga(4)L(6)](12-) (1; L = 1,5-bis[2,3-dihydroxybenzamido]naphthalene) can act as a molecular host in aqueous solution and bind cationic guest molecules to its highly charged exterior surface or within its hydrophobic interior cavity. The distinct internal cavity of host 1 modifies the physical properties and reactivity of bound guest molecules and can be used to catalyze a variety of chemical transformations. Noncovalent host-guest interactions in large part control guest binding, molecular recognition and the chemical reactivity of bound guests. Herein we examine equilibrium isotope effects (EIEs) on both exterior and interior guest binding to host 1 and use these effects to probe the details of noncovalent host-guest interactions. For both interior and exterior binding of a benzylphosphonium guest in aqueous solution, protiated guests are found to bind more strongly to host 1 (K(H)/K(D) > 1) and the preferred association of protiated guests is driven by enthalpy and opposed by entropy. Deuteration of guest methyl and benzyl C-H bonds results in a larger EIE than deuteration of guest aromatic C-H bonds. The observed EIEs can be well explained by considering changes in guest vibrational force constants and zero-point energies. DFT calculations further confirm the origins of these EIEs and suggest that changes in low-frequency guest C-H/D vibrational motions (bends, wags, etc.) are primarily responsible for the observed EIEs. © 2011 American Chemical Society

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

PubMed

Kellner, Julian N; Reinstein, Jochen; Meinhart, Anton

2015-03-11

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Investigation of the interaction between berberine and nucleosomes in solution: Spectroscopic and equilibrium dialysis approach

NASA Astrophysics Data System (ADS)

Rabbani-Chadegani, Azra; Mollaei, Hossein; Sargolzaei, Javad

2017-02-01

Berberine is a natural plant alkaloid with high pharmacological potential. Although its interaction with free DNA has been the subject of several reports, to date there is no work concerning the effect of berberine on nucleoprotein structure of DNA, the nucleosomes. The present study focuses on the binding affinity of berberine to nucleosomes and histone H1 employing various spectroscopic techniques, fluorescence, circular dichroism, thermal denaturation as well as equilibrium dialysis. The results showed that the binding of berberine to nucleosomes is positive cooperative with Ka = 5.57 × 103 M- 1. Berberine quenched with the chromophores of protein moiety of nucleosomes and reduced fluorescence emission intensity at 335 nm with Ksv value of 0.135. Binding of berberine to nucleosomes decreased the absorbance at 210 and 260 nm, produced hypochromicity in thermal denaturation profiles and its affinity to nucleoprotein structure of nucleosomes was much higher than to free DNA. Berberine also exhibited high affinity to histone H1 in solution and the binding was positive cooperative with. Ka = 3.61 × 103 M- 1. Moreover berberine decreased fluorescence emission intensity of H1 by quenching with tyrosine residue in its globular core domain. The circular dichroism profiles demonstrated that the binding of drug induced secondary structural changes in both DNA stacking and histone H1. It is concluded that berberine is genotoxic drug, interacts with nucleosomes and in this process histone H1 is involved to exert its anticancer activity.

Plasma binding of an alpha-blocking agent, nicergoline--affinity for serum albumin and native and modified alpha 1-acid glycoprotein.

PubMed

Robert, L; Migne, J; Santonja, R; Zini, R; Schmid, K; Tillement, J P

1983-06-01

The binding of nicergoline, an alpha-blocking drug, by human plasma proteins was studied using gel filtration, polyacrylamide gel electrophoresis, and equilibrium dialysis techniques. 3H-labeled nicergoline added to plasma was eluted together with two major protein fractions, one containing mainly serum albumin, the other glycoproteins such as alpha 1-acid glycoprotein (alpha 1-AG). Equilibrium dialysis experiments with pure human serum albumin and alpha 1-AG as well as with its chemically modified forms, desialylated, carboxymethylated, and both desialylated and carboxymethylated alpha 1-AG gave the following results: nicergoline has about a 4-fold higher affinity for alpha 1-AG than for serum albumin. There are two binding sites per molecule on serum albumin and one on alpha 1-AG. The binding parameters of alpha 1-AG were not significantly modified by desialylation or carboxymethylation. Only desialylated and carboxymethylated alpha 1-AG showed a decreased binding for nicergoline, suggesting conformational modifications induced by these combined treatments. The fact that desialylated alpha 1-AG keeps its affinity for nicergoline suggests the possibility of a selective introduction of this drug in cells possessing the Ashwell-type specific receptor for desialylated alpha 1-AG, for instance hepatocytes. Increased serum alpha 1-AG concentration induced by inflammatory reactions will also modify the distribution of bound nicergoline between serum albumin and alpha 1-AG and as a consequence its half-life and cell distribution.

Validation of a simple method of estimating plasma free cortisol: role of cortisol binding to albumin.

PubMed

Dorin, Richard I; Pai, Hemanth K; Ho, Jui T; Lewis, John G; Torpy, David J; Urban, Frank K; Qualls, Clifford R

2009-01-01

To develop, optimize, and validate a generalized mass action, equilibrium solution that incorporates measured concentrations of albumin as well as cortisol binding globulin (CBG) to estimate free cortisol. Free cortisol was estimated by Coolens method or by cubic equilibrium equation and compared to measured free cortisol, determined by ultrafiltration method, in subjects with septic shock (n=45), sepsis (n=19), and healthy controls (n=10) at 0, 30, and 60 min following administration of cosyntropin (250 mcg). The data set also included repeat testing in 30 subjects following recovery from sepsis/septic shock. The equilibrium dissociation constant for cortisol binding to albumin (K(A)) was optimized by non-linear regression. The cubic equilibrium solution was also used to model the influence of cortisol, CBG, and albumin concentration on free cortisol. Compared to measured free cortisol, the cubic solution, using an optimized K(A) of 137,800 nM, was less biased than Coolens solution, with mean percent error of -23.0% vs. -41.1% (paired t test, P<0.001). Standard deviation values were also significantly lower (Wilks' test, P<0.001) for the cubic solution (SD 35.8% vs. 40.8% for cubic vs. Coolens, respectively). Modeling studies using the cubic solution suggest an interaction effect by which low concentrations of CBG and albumin contribute to a greater increase in free cortisol than the sum of their independent effects. Mass action solutions that incorporate the measured concentration of albumin as well as CBG provide a reasonably accurate estimate of free cortisol that generalizes to conditions of health as well as a setting of hypercortisolism and low CBG and albumin concentrations associated with septic shock. Modeling studies emphasize the significant contribution of albumin deficiency and albumin-bound cortisol under conditions of CBG-deficiency, and identify a synergistic effect by which combined CBG and albumin deficiency contribute to elevation of free cortisol in septic shock.

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

PubMed Central

Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

1975-01-01

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

PubMed

Wright, J F; Pernollet, M; Reboul, A; Aude, C; Colomb, M G

1992-05-05

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

Exploring transition pathway and free-energy profile of large-scale protein conformational change by combining normal mode analysis and umbrella sampling molecular dynamics.

PubMed

Wang, Jinan; Shao, Qiang; Xu, Zhijian; Liu, Yingtao; Yang, Zhuo; Cossins, Benjamin P; Jiang, Hualiang; Chen, Kaixian; Shi, Jiye; Zhu, Weiliang

2014-01-09

Large-scale conformational changes of proteins are usually associated with the binding of ligands. Because the conformational changes are often related to the biological functions of proteins, understanding the molecular mechanisms of these motions and the effects of ligand binding becomes very necessary. In the present study, we use the combination of normal-mode analysis and umbrella sampling molecular dynamics simulation to delineate the atomically detailed conformational transition pathways and the associated free-energy landscapes for three well-known protein systems, viz., adenylate kinase (AdK), calmodulin (CaM), and p38α kinase in the absence and presence of respective ligands. For each protein under study, the transient conformations along the conformational transition pathway and thermodynamic observables are in agreement with experimentally and computationally determined ones. The calculated free-energy profiles reveal that AdK and CaM are intrinsically flexible in structures without obvious energy barrier, and their ligand binding shifts the equilibrium from the ligand-free to ligand-bound conformation (population shift mechanism). In contrast, the ligand binding to p38α leads to a large change in free-energy barrier (ΔΔG ≈ 7 kcal/mol), promoting the transition from DFG-in to DFG-out conformation (induced fit mechanism). Moreover, the effect of the protonation of D168 on the conformational change of p38α is also studied, which reduces the free-energy difference between the two functional states of p38α and thus further facilitates the conformational interconversion. Therefore, the present study suggests that the detailed mechanism of ligand binding and the associated conformational transition is not uniform for all kinds of proteins but correlated to their respective biological functions.

Isolation and characterization of a new zinc-binding protein from albacore tuna plasma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dyke, B.; Hegenauer, J.; Saltman, P.

1987-06-02

The protein responsible for sequestering high levels of zinc in the plasma of the albacore tuna (Thunnus alalunga) has been isolated by sequential chromatography. The glycoprotein has a molecular weight of 66,000. Approximately 8.2% of its amino acid residues are histidines. Equilibrium dialysis experiments show it to bind 3 mol of zinc/mol of protein. The stoichiometric constant for the association of zinc with a binding site containing three histidines was determined to be 10/sup 9.4/. This protein is different from albumin and represents a previously uncharacterized zinc transport protein.

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators

DOE PAGES

Ahmad, Md. Faiz; Huff, Sarah E.; Pink, John; ...

2015-10-21

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC 50s and K Ds while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive modemore » of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Finally, together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.« less

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

Induced Fit in Protein Multimerization: The HFBI Case

PubMed Central

Riccardi, Laura

2016-01-01

Hydrophobins, produced by filamentous fungi, are small amphipathic proteins whose biological functions rely on their unique surface-activity properties. Understanding the mechanistic details of the multimerization process is of primary importance to clarify the interfacial activity of hydrophobins. We used free energy calculations to study the role of a flexible β-hairpin in the multimerization process in hydrophobin II from Trichoderma reesei (HFBI). We characterized how the displacement of this β-hairpin controls the stability of the monomers/dimers/tetramers in solution. The regulation of the oligomerization equilibrium of HFBI will necessarily affect its interfacial properties, fundamental for its biological function and for technological applications. Moreover, we propose possible routes for the multimerization process of HFBI in solution. This is the first case where a mechanism by which a flexible loop flanking a rigid patch controls the protein-protein binding equilibrium, already known for proteins with charged binding hot-spots, is described within a hydrophobic patch. PMID:27832079

Visualizing the orientational dependence of an intermolecular potential

NASA Astrophysics Data System (ADS)

Sweetman, Adam; Rashid, Mohammad A.; Jarvis, Samuel P.; Dunn, Janette L.; Rahe, Philipp; Moriarty, Philip

2016-02-01

Scanning probe microscopy can now be used to map the properties of single molecules with intramolecular precision by functionalization of the apex of the scanning probe tip with a single atom or molecule. Here we report on the mapping of the three-dimensional potential between fullerene (C60) molecules in different relative orientations, with sub-Angstrom resolution, using dynamic force microscopy (DFM). We introduce a visualization method which is capable of directly imaging the variation in equilibrium binding energy of different molecular orientations. We model the interaction using both a simple approach based around analytical Lennard-Jones potentials, and with dispersion-force-corrected density functional theory (DFT), and show that the positional variation in the binding energy between the molecules is dominated by the onset of repulsive interactions. Our modelling suggests that variations in the dispersion interaction are masked by repulsive interactions even at displacements significantly larger than the equilibrium intermolecular separation.

Modeling chain folding in protein-constrained circular DNA.

PubMed Central

Martino, J A; Olson, W K

1998-01-01

An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems. PMID:9591675

Experimental measurement of binding energy, selectivity, and allostery using fluctuation theorems.

PubMed

Camunas-Soler, Joan; Alemany, Anna; Ritort, Felix

2017-01-27

Thermodynamic bulk measurements of binding reactions rely on the validity of the law of mass action and the assumption of a dilute solution. Yet, important biological systems such as allosteric ligand-receptor binding, macromolecular crowding, or misfolded molecules may not follow these assumptions and may require a particular reaction model. Here we introduce a fluctuation theorem for ligand binding and an experimental approach using single-molecule force spectroscopy to determine binding energies, selectivity, and allostery of nucleic acids and peptides in a model-independent fashion. A similar approach could be used for proteins. This work extends the use of fluctuation theorems beyond unimolecular folding reactions, bridging the thermodynamics of small systems and the basic laws of chemical equilibrium. Copyright © 2017, American Association for the Advancement of Science.

Quantifying Na(I)-insulin and K(I)-insulin non-covalent complexes by ESI-MS method and calculation of their equilibrium constants.

PubMed

Gülfen, Mustafa; Özdemir, Abdil; Lin, Jung-Lee; Chen, Chung-Hsuan

2017-10-01

In this study, the dissociation and formation equilibrium constants of Na(I)-insulin and K(I)-insulin complexes have been calculated after the quantifying them on ESI mass spectrometer. The ESI-MS spectra of the complexes were measured by using the solvents as 50% MeOH in water and 100% water. The effect of pH on the Na(I)-insulin and K(I)-insulin complex formation were examined. Serial binding of Na(I) and K(I) ions to the insulin molecule were observed in the ESI-MS measurements. The first formation equilibrium constants were calculated as K f1 : 5.48×10 3 1/M for Na(I)-insulin complex and K f1 : 4.87×10 3 1/M for K(I)-insulin in water. The binding capability of Na(I) ions to insulin molecule is higher than the capability of K(I) ions. In case of a comparison together with Ca(II)-insulin and Mg(II)-insulin, the formation equilibrium constants (K f1 ) are in order of Ca(II)-insulin>Mg(II)-insulin>Na(I)-insulin>K(I)-insulin in water. The results showed that Na(I) and K(I) ions are involved in the formation of the non-covalent complexes with insulin molecule, since high extracellular and intracellular concentrations of them in the body. Copyright © 2017 Elsevier B.V. All rights reserved.

Thermodynamics of Biological Processes

PubMed Central

Garcia, Hernan G.; Kondev, Jane; Orme, Nigel; Theriot, Julie A.; Phillips, Rob

2012-01-01

There is a long and rich tradition of using ideas from both equilibrium thermodynamics and its microscopic partner theory of equilibrium statistical mechanics. In this chapter, we provide some background on the origins of the seemingly unreasonable effectiveness of ideas from both thermodynamics and statistical mechanics in biology. After making a description of these foundational issues, we turn to a series of case studies primarily focused on binding that are intended to illustrate the broad biological reach of equilibrium thinking in biology. These case studies include ligand-gated ion channels, thermodynamic models of transcription, and recent applications to the problem of bacterial chemotaxis. As part of the description of these case studies, we explore a number of different uses of the famed Monod–Wyman–Changeux (MWC) model as a generic tool for providing a mathematical characterization of two-state systems. These case studies should provide a template for tailoring equilibrium ideas to other problems of biological interest. PMID:21333788

Lactoferrin-binding proteins in Shigella flexneri.

PubMed Central

Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S

1992-01-01

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403

Screening lactic acid bacteria strains with ability to bind di-n-butyl phthalate via Turbiscan technique.

PubMed

Lili, Zhao; Hongfei, Zhao; Shoukat, Sana; Xiaochen, Zhang; Bolin, Zhang

2017-06-01

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant that poses a risk to humans. Previous work indicates that the ability of lactic acid bacteria (LAB) to bind phthalic acid esters is strain-specific. As cell suspensions of LAB strains in aqueous solution are likely to be colloidal dispersions, this study provided a technique to efficiently screen LAB strains that bind DBP via Turbiscan, which has been widely used to measure the stability of emulsions or colloidal dispersions. Eleven LAB strains belonging to Lactobacillus plantarum, Lb. pentosus, Lb. paralimentarius, Lb. helveticus, Leuconostoc mesenteroides, Lb. acidophilus, Bifidobacterium lactis, and Bifidobacterium bifidum species were used in this study, and seven of them were selected to test in an earlier stage of exploring the process for finding a screening method; others were used for a validation test. It was observed that the various values of the 10 h Turbiscan Stability Index (TSI) of the cell suspension from each strain, at the equilibrium time of dispersed particles according to the peak thickness of cell-suspensions as measured by Turbiscan, had significant negative correlations with the DBP-binding percentage of LAB strains. Higher TSI values are correlated with lower binding of bacteria strains to DBP with a correlation coefficient of 0.8292. Cell surface hydrocarbons of LAB strains and their adherence were observed to correlate with DBP-binding percentages and may lead to the different states of aggregation or equilibrium of bacterial cell-suspensions, and the aggregation of bacterial cells resulted in fewer binding sites in the cell wall for DBP. Finally, four LAB strains were randomly selected to verify the feasibility of the method. In all, the findings demonstrate that TSI might be used as a tool to quickly screen strains that bind DBP. The present work could be extended to the removal of other toxic compounds, when screening of high-efficiency strains is required.

Conformational Plasticity of the Cell-Penetrating Peptide SAP As Revealed by Solid-State 19F-NMR and Circular Dichroism Spectroscopies.

PubMed

Afonin, Sergii; Kubyshkin, Vladimir; Mykhailiuk, Pavel K; Komarov, Igor V; Ulrich, Anne S

2017-07-13

The cell-penetrating peptide SAP, which was designed as an amphipathic poly-l-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state 19 F-NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. Trifluoromethyl-bicyclopentyl-glycine (CF 3 -Bpg) and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF 3 -MePro) were used as labels for 19 F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF 3 -MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and 19 F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered "raft"-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a nonaggregated state was structured as a mixture of nonideal extended conformations reflecting the equilibrium already present in solution, i.e., before binding to the membrane.

Exploring the Stability of Ligand Binding Modes to Proteins by Molecular Dynamics Simulations: A Cross-docking Study.

PubMed

Liu, Kai; Kokubo, Hironori

2017-10-23

Docking has become an indispensable approach in drug discovery research to predict the binding mode of a ligand. One great challenge in docking is to efficiently refine the correct pose from various putative docking poses through scoring functions. We recently examined the stability of self-docking poses under molecular dynamics (MD) simulations and showed that equilibrium MD simulations have some capability to discriminate between correct and decoy poses. Here, we have extended our previous work to cross-docking studies for practical applications. Three target proteins (thrombin, heat shock protein 90-alpha, and cyclin-dependent kinase 2) of pharmaceutical interest were selected. Three comparable poses (one correct pose and two decoys) for each ligand were then selected from the docking poses. To obtain the docking poses for the three target proteins, we used three different protocols, namely: normal docking, induced fit docking (IFD), and IFD against the homology model. Finally, five parallel MD equilibrium runs were performed on each pose for the statistical analysis. The results showed that the correct poses were generally more stable than the decoy poses under MD. The discrimination capability of MD depends on the strategy. The safest way was to judge a pose as being stable if any one run among five parallel runs was stable under MD. In this case, 95% of the correct poses were retained under MD, and about 25-44% of the decoys could be excluded by the simulations for all cases. On the other hand, if we judge a pose as being stable when any two or three runs were stable, with the risk of incorrectly excluding some correct poses, approximately 31-53% or 39-56% of the two decoys could be excluded by MD, respectively. Our results suggest that simple equilibrium simulations can serve as an effective filter to exclude decoy poses that cannot be distinguished by docking scores from the computationally expensive free-energy calculations.

Modeling of the solution interaction properties of plastic materials used in pharmaceutical product container systems.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy; Sadain, Salma

2009-01-01

Material/water equilibrium binding constants (Eb) were determined for 14 organic solutes and 17 plastic raw materials that could be used in pharmaceutical product container systems. Correlations between the measured binding constants and the organic solute's octanol/water and hexane/water partition coefficients were obtained. In general, while the materials examined exhibited a wide range of binding characteristics, the tested materials by and large fell within two broad classes: (1) those that were octanol-like in their binding characteristics, and (2) those that were hexane-like. Materials of the same class (e.g., polypropylenes) generally had binding models that were very similar. Rank ordering of the materials in terms of their magnitude of drug binding (least binding to most binding) was as follows: polypropylene < polyethylene < polyamide < styrene-ethylene-butylene-styrene < copolyester ether elastomer approximately equal to amine-terminated poly fatty acid amide polymer. The utilization of the developed models to estimate drug loss via sorption by the container is discussed.

Effect of enzymatic deamidation of soy protein by protein-glutaminase on the flavor-binding properties of the protein under aqueous conditions.

PubMed

Suppavorasatit, Inthawoot; Cadwallader, Keith R

2012-08-15

The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding.

Biosorption of trivalent chromium on the brown seaweed biomass.

PubMed

Yun, Y S; Park, D; Park, J M; Volesky, B

2001-11-01

Biosorption has attracted attention as a cost-effective means for the treatment of metal-bearing wastewater. However, the mechanism of metal binding is not clearly understood, and consequently, modeling of the biosorption performance is still raising debates. In this study, the biosorption of trivalent chromium was investigated with protonated brown alga Ecklonia biomass as a model system. Titration of the biomass revealed that it contains at least three types of functional groups. The Fourier transform infrared spectrometry showed that the carboxyl group was the chromium-binding site within the pH range (pH 1-5) used in this study, where chromium does not precipitate. The pK value and the number of carboxyl groups were estimated to be 4.6 +/- 0.1 and 2.2 +/- 0.1 mmol/g, respectively. The equilibrium sorption isotherms determined at different solution pH indicated that the uptake of chromium increased significantly with increasing pH. A model for the description of chromium biosorption was developed incorporating the hydrolysis reactions that chromium undergoes in the aquatic phase. The model was able to predict the equilibrium sorption experimental data at different pH values and chromium concentrations. In addition, the speciation of the binding site as a function of the solution pH was predicted using the model in order to visualize the distribution of chromium ionic species on the binding site.

Thermodynamics of impurity-enhanced vacancy formation in metals

NASA Astrophysics Data System (ADS)

Bukonte, Laura; Ahlgren, Tommy; Heinola, Kalle

2017-01-01

Hydrogen induced vacancy formation in metals and metal alloys has been of great interest during the past couple of decades. The main reason for this phenomenon, often referred to as the superabundant vacancy formation, is the lowering of vacancy formation energy due to the trapping of hydrogen. By means of thermodynamics, we study the equilibrium vacancy formation in fcc metals (Pd, Ni, Co, and Fe) in correlation with the H amounts. The results of this study are compared and found to be in good agreement with experiments. For the accurate description of the total energy of the metal-hydrogen system, we take into account the binding energies of each trapped impurity, the vibrational entropy of defects, and the thermodynamics of divacancy formation. We demonstrate the effect of vacancy formation energy, the hydrogen binding, and the divacancy binding energy on the total equilibrium vacancy concentration. We show that the divacancy fraction gives the major contribution to the total vacancy fraction at high H fractions and cannot be neglected when studying superabundant vacancies. Our results lead to a novel conclusion that at high hydrogen fractions, superabundant vacancy formation takes place regardless of the binding energy between vacancies and hydrogen. We also propose the reason of superabundant vacancy formation mainly in the fcc phase. The equations obtained within this work can be used for any metal-impurity system, if the impurity occupies an interstitial site in the lattice.

Theory for rates, equilibrium constants, and Brønsted slopes in F1-ATPase single molecule imaging experiments

PubMed Central

Volkán-Kacsó, Sándor; Marcus, Rudolph A.

2015-01-01

A theoretical model of elastically coupled reactions is proposed for single molecule imaging and rotor manipulation experiments on F1-ATPase. Stalling experiments are considered in which rates of individual ligand binding, ligand release, and chemical reaction steps have an exponential dependence on rotor angle. These data are treated in terms of the effect of thermodynamic driving forces on reaction rates, and lead to equations relating rate constants and free energies to the stalling angle. These relations, in turn, are modeled using a formalism originally developed to treat electron and other transfer reactions. During stalling the free energy profile of the enzymatic steps is altered by a work term due to elastic structural twisting. Using biochemical and single molecule data, the dependence of the rate constant and equilibrium constant on the stall angle, as well as the Børnsted slope are predicted and compared with experiment. Reasonable agreement is found with stalling experiments for ATP and GTP binding. The model can be applied to other torque-generating steps of reversible ligand binding, such as ADP and Pi release, when sufficient data become available. PMID:26483483

Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I.

PubMed

Hoggett, J G; Kellett, G L

1995-01-15

Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state.

Programming A Molecular Relay for Ultrasensitive Biodetection through 129 Xe NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanfei; Roose, Benjamin W.; Philbin, John P.

2015-12-21

We reported a supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized 129Xe NMR. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. Moreover, the protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn 2+ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidinmore » by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive 129Xe NMR spectroscopy.« less

Binding of warfarin influences the acid-base equilibrium of H242 in sudlow site I of human serum albumin.

PubMed

Perry, Jennifer L; Goldsmith, Michael R; Williams, T Richard; Radack, Kyle P; Christensen, Trine; Gorham, Justin; Pasquinelli, Melissa A; Toone, Eric J; Beratan, David N; Simon, John D

2006-01-01

Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W-) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 x 10(5) M(-1). ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W- is accompanied by uptake of 0.30+/-0.02 protons from the solvent. This measurement suggests that the binding of W- is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W-.

Graphene-Based Liquid-Gated Field Effect Transistor for Biosensing: Theory and Experiments

PubMed Central

Reiner-Rozman, Ciril; Larisika, Melanie; Nowak, Christoph; Knoll, Wolfgang

2015-01-01

We present an experimental and theoretical characterization for reduced Graphene-Oxide (rGO) based FETs used for biosensing applications. The presented approach shows a complete result analysis and theoretically predictable electrical properties. The formulation was tested for the analysis of the device performance in the liquid gate mode of operation with variation of the ionic strength and pH-values of the electrolytes in contact with the FET. The dependence on the Debye length was confirmed experimentally and theoretically, utilizing the Debye length as a working parameter and thus defining the limits of applicability for the presented rGO-FETs. Furthermore, the FETs were tested for the sensing of biomolecules (bovine serum albumin (BSA) as reference) binding to gate-immobilized anti-BSA antibodies and analyzed using the Langmuir binding theory for the description of the equilibrium surface coverage as a function of the bulk (analyte) concentration. The obtained binding coefficients for BSA are found to be same as in results from literature, hence confirming the applicability of the devices. The FETs used in the experiments were fabricated using wet-chemically synthesized graphene, displaying high electron and hole mobility (μ) and provide the strong sensitivity also for low potential changes (by change of pH, ion concentration, or molecule adsorption). The binding coefficient for BSA-anti-BSA interaction shows a behavior corresponding to the Langmuir adsorption theory with a Limit of Detection (LOD) in the picomolar concentration range. The presented approach shows high reproducibility and sensitivity and a good agreement of the experimental results with the calculated data. PMID:25791463

Analytical RISM-MP2 free energy gradient method: Application to the Schlenk equilibrium of Grignard reagent

NASA Astrophysics Data System (ADS)

Mori, Toshifumi; Kato, Shigeki

2007-03-01

We present a method to evaluate the analytical gradient of reference interaction site model Møller-Plesset second order free energy with respect to solute nuclear coordinates. It is applied to calculate the geometries and energies in the equilibria of the Grignard reagent (CH 3MgCl) in dimethylether solvent. The Mg-Mg and Mg-Cl distances as well as the binding energies of solvents are largely affected by the dynamical electron correlation. The solvent effect on the Schlenk equilibrium is examined.

The synthesis and characterization of environmentally-responsive water-swellable and water-soluble polymers for wastewater remediation

NASA Astrophysics Data System (ADS)

Armentrout, Rodney Scott

The primary research goal is the development of new polymeric materials that demonstrate the environmentally-responsive sequestration of common water foulants, including surfactants and oils. Water-swellable and water-soluble polymers have been synthesized, structurally characterized, and their physical properties have been determined. In addition, the ability of the materials to sequester model water foulants has been evaluated. Anionic crosslinked polymer networks of 2-acrylamido-2-methyl-1-propanesulfonic acid, acrylamide, and methylene bisacrylamide have been synthesized and characterized by determining the equilibrium water contents as a function of ionic content of the polymer network. The molar ratio of bound surfactant to ionic group was determined to be less than one for all hydrogels studied, indicating an ion-exchange binding mechanism with minimal hydrophobic interactions between bound and unbound surfactant molecules is responsible for surfactant binding. Cationic crosslinked cyclopolymer networks of N,N-diallyl- N-methyl amine (DAMA) and N,N,N,N-tetraallyl ammonium chloride (TAAC) have been synthesized and characterized by determining the equilibrium water content as a function of pH. A maximum in the equilibrium water content is observed for pH-6 when the polymer is fully ionized. The solubilization of a model water foulant, p-cresol, by the polymeric surfactant, Pluronic F127, has been studied via equilibrium dialysis, dynamic light scattering and ultrafiltration experiments. It has been shown that at 25°C p-cresol is readily solubilized by F127 since the polymeric surfactant exists in a multimer conformation. Ultrafiltration experiments have demonstrated that the polymer-foulant binding interactions are largely unaffected by shear in a hollow fiber membrane. Copolymers of the zwitterionic monomer, 3-(N,N-diallyl- N-methyl ammonio) propane sulfonate (DAMAPS) and N,N-diallyl- N,N-dimethylammonium chloride (DADMAC) (the DADS series) or the pH-responsive hydrophobic monomer, N,N-diallyl-N-methyl amine (DAMA) (the DAMS series) have been prepared in a 0.5 M NaCl aqueous solution using 2-hydroxy-1-[4-(hydroxy-ethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959) as the free-radical photoinitiator. 13C NMR data indicate that the resulting polymers maintain the five-membered ring structure in the cis conformation common to diallylammonium salts. Equilibrium dialysis experiments demonstrate that pH-responsive hydrophobic microdomain formation may be utilized to control the solubilization of the organic solute, p-cresol. Ultrafiltration experiments have demonstrated that the polymer-foulant binding interactions are largely unaffected by shear in a hollow fiber membrane. Macromolecular aggregates of the poly( N,N-diallyl-N-methyl amine)/p-cresol complexes lead to fouling of the ultrafiltration membrane. However, incorporation of the sulfobetaine moiety hinders the formation of the macroscopic structures and higher permeate flux rates are achieved. (Abstract shortened by UMI.)

Iodination of (Tyr11)somatostatin yields a super high affinity ligand for somatostatin receptors in GH4C1 pituitary cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Presky, D.H.; Schonbrunn, A.

1988-11-01

GH4C1 cells are a clonal strain of rat pituitary tumor cells which contain high affinity receptors for the inhibitory neuropeptide somatostatin (SRIF). In contrast to other peptides that bind to specific receptors on these cells, receptor-bound (125I-Tyr1)SRIF does not undergo rapid endocytosis. Rather, partial degradation to 125I-tyrosine occurs concomitantly with the dissociation of (125I-Tyr1)SRIF from cell surface receptors. In this study we characterize the binding, biological activity and receptor-mediated degradation of (125I-Tyr11)SRIF, a SRIF analog that is radiolabeled in the center of the molecule. The binding of trace concentrations of (125I-Tyr11)SRIF (less than 50 pM) required 6 hr to reachmore » equilibrium at 37 degrees compared with the 60 min required for (125I-Tyr1)SRIF. Analysis of the kinetics of (125I- Tyr11)SRIF binding showed that the rate constant for association (kon = 1.7 x 10(8) M-8min-1) was similar to that for (125I-Tyr1)SRIF (0.8 x 10(8) M-1min-1). However, the two radioligands exhibited markedly different dissociation kinetics; the koff for (125I-Tyr11)SRIF was 0.002 min-1 compared with the value of 0.02 min-1 for (125I-Tyr1) SRIF. In agreement with its much slower rate of dissociation, (125I-Tyr11)SRIF bound to the SRIF receptor with higher affinity (Kd = 70 pM) than did (125I-Tyr1)SRIF (Kd = 350 pM). However, the apparent ED50 for (I-Tyr11)SRIF to inhibit cAMP accumulation (1.9 +/- 0.4 nM) was greater than the ED50 for SRIF (0.19 +/- 0.04 nM). The low potency of (I-Tyr11)SRIF probably resulted from the fact that subsaturating concentrations of this peptide did not achieve equilibrium binding during the 30-min incubation used to assay biological activity. As previously reported for (125I-Tyr1)SRIF, receptor-bound (125I-Tyr11)SRIF was not internalized and was released from the cells as a mixture of intact (125I-Tyr11)SRIF (30%) and the degradation product 125I-tyrosine (65%).« less

Replica exchange molecular dynamics simulations provide insight into substrate recognition by small heat shock proteins.

PubMed

Patel, Sunita; Vierling, Elizabeth; Tama, Florence

2014-06-17

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

PubMed Central

Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radić, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

2008-01-01

Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 Å in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids. PMID:18477694

Molecular dynamics simulation of the Escherichia coli NikR protein: equilibrium conformational fluctuations reveal interdomain allosteric communication pathways.

PubMed

Bradley, Michael J; Chivers, Peter T; Baker, Nathan A

2008-05-16

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

Molecular dynamics simulation of the Escherichia coli NikR protein: Equilibrium conformational fluctuations reveal inter-domain allosteric communication pathways

PubMed Central

Bradley, Michael J.; Chivers, Peter T.; Baker, Nathan A.

2008-01-01

Summary E. coliNikR is a homotetrameric Ni2+- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of 2 distinct domains. The N-terminal fifty amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The eighty-three residue C-terminal nickel-binding domain forms an ACT-fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics (MD) simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni on DNA-binding activity. The molecular simulation data was analyzed using two different correlation measures based on fluctuations in atomic position and non-covalent contacts, together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni2+ and DNA binding sites, which are separated by 40 Å. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains. PMID:18433769

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Synthesis and characterization of DNA minor groove binding alkylating agents.

PubMed

Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

2013-01-18

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy

PubMed Central

Lo, Kai-Yin; Sun, Yung-Shin; Landry, James P.; Zhu, Xiangdong; Deng, Wenbin

2012-01-01

Conventional fluorescent microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in ways often uncharacterized. The binding between antibody and antigen might not be in the native situation after such conjugation. Here, we present an oblique-incidence reflectivity difference (OI-RD) microscope as an effective method for label-free, real-time detection of cell surface markers and apply such a technique to analysis of Stage-Specific Embryonic Antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies only bind to the surface of the stem cells while not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. The results concluded that OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells. The information could be another indicator to determine the cell stages. PMID:21781038

Differential Binding Models for Direct and Reverse Isothermal Titration Calorimetry.

PubMed

Herrera, Isaac; Winnik, Mitchell A

2016-03-10

Isothermal titration calorimetry (ITC) is a technique to measure the stoichiometry and thermodynamics from binding experiments. Identifying an appropriate mathematical model to evaluate titration curves of receptors with multiple sites is challenging, particularly when the stoichiometry or binding mechanism is not available. In a recent theoretical study, we presented a differential binding model (DBM) to study calorimetry titrations independently of the interaction among the binding sites (Herrera, I.; Winnik, M. A. J. Phys. Chem. B 2013, 117, 8659-8672). Here, we build upon our DBM and show its practical application to evaluate calorimetry titrations of receptors with multiple sites independently of the titration direction. Specifically, we present a set of ordinary differential equations (ODEs) with the general form d[S]/dV that can be integrated numerically to calculate the equilibrium concentrations of free and bound species S at every injection step and, subsequently, to evaluate the volume-normalized heat signal (δQ(V) = δq/dV) of direct and reverse calorimetry titrations. Additionally, we identify factors that influence the shape of the titration curve and can be used to optimize the initial concentrations of titrant and analyte. We demonstrate the flexibility of our updated DBM by applying these differentials and a global regression analysis to direct and reverse calorimetric titrations of gadolinium ions with multidentate ligands of increasing denticity, namely, diglycolic acid (DGA), citric acid (CIT), and nitrilotriacetic acid (NTA), and use statistical tests to validate the stoichiometries for the metal-ligand pairs studied.

A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.

PubMed

Wiley, J S; Brocklebank, A M; Snook, M B; Jamieson, G P; Sawyer, W H; Craik, J D; Cass, C E; Robins, M J; McAdam, D P; Paterson, A R

1991-02-01

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Mechanism of pKID/KIX Association Studied by Molecular Dynamics Free Energy Simulations.

PubMed

Bomblies, Rainer; Luitz, Manuel P; Zacharias, Martin

2016-08-25

The phosphorylated kinase-inducible domain (pKID) associates with the kinase interacting domain (KIX) via a coupled folding and binding mechanism. The pKID domain is intrinsically disordered when unbound and upon phosphorylation at Ser133 binds to the KIX domain adopting a well-defined kinked two-helix structure. In order to identify putative hot spot residues of binding that could serve as an initial stable anchor, we performed in silico alanine scanning free energy simulations. The simulations indicate that charged residues including the phosphorylated central Ser133 of pKID make significant contributions to binding. However, these are of slightly smaller magnitude compared to several hydrophobic side chains not defining a single dominant binding hot spot. Both continuous molecular dynamics (MD) simulations and free energy analysis demonstrate that phosphorylation significantly stabilizes the central kinked motif around Ser133 of pKID and shifts the conformational equilibrium toward the bound conformation already in the absence of KIX. This result supports a view that pKID/KIX association follows in part a conformational selection process. During a 1.5 μs explicit solvent MD simulation, folding of pKID on the surface of KIX was observed after an initial contact at the bound position of the phosphorylation site was enforced following a sequential process of αA helix association and a stepwise association and folding of the second αB helix compatible with available experimental results.

Allosteric Modulation of protein oligomerization: an emerging approach to drug design

NASA Astrophysics Data System (ADS)

Gabizon, Ronen; Friedler, Assaf

2014-03-01

Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the "shiftides" approach developed in our lab.

Accelerated Self-Replication under Non-Equilibrium, Periodic Energy Delivery

NASA Astrophysics Data System (ADS)

Zhang, Rui; Olvera de La Cruz, Monica

2014-03-01

Self-replication is a remarkable phenomenon in nature that has fascinated scientists for decades. In a self-replicating system, the original units are attracted to a template, which induce their binding. In equilibrium, the energy required to disassemble the newly assembled copy from the mother template is supplied by thermal energy. The possibility of optimizing self-replication is explored by controlling the frequency at which energy is supplied to the system. A model system inspired by a class of light switchable colloids is considered where light is used to control the interactions. Conditions under which self-replication can be significantly more effective under non-equilibrium, cyclic energy delivery than under equilibrium constant energy conditions are identified. Optimal self-replication does not require constant energy expenditure. Instead, the proper timing at which energy is delivered to the system is an essential controllable parameter to induce high replication rates. This work was supported by the Non-Equilibrium Energy Research Center (NERC), which is an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0000989.

Dye-induced aggregation of single stranded RNA: a mechanistic approach.

PubMed

Biver, Tarita; Ciatto, Carlo; Secco, Fernando; Venturini, Marcella

2006-08-15

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.

A New Model for Allosteric Regulation of Phenylalanine Hydroxylase: Implications for Disease and Therapeutics

PubMed Central

Stith, Linda; Lawrence, Sarah H.; Andrake, Mark; Dunbrack, Roland L.

2013-01-01

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ~90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the inter-subunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic. PMID:23296088

Apo and InsP[subscript 3]-bound crystal structures of the ligand-binding domain of an InsP[subscript 3] receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chun-Chi; Baek, Kyuwon; Lu, Zhe

2012-05-08

We report the crystal structures of the ligand-binding domain (LBD) of a rat inositol 1,4,5-trisphosphate receptor (InsP{sub 3}R) in its apo and InsP{sub 3}-bound conformations. Comparison of these two conformations reveals that LBD's first {beta}-trefoil fold ({beta}-TF1) and armadillo repeat fold (ARF) move together as a unit relative to its second {beta}-trefoil fold ({beta}-TF2). Whereas apo LBD may spontaneously transition between gating conformations, InsP{sub 3} binding shifts this equilibrium toward the active state.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

High-throughput analysis using non-depletive SPME: challenges and applications to the determination of free and total concentrations in small sample volumes.

PubMed

Boyacı, Ezel; Bojko, Barbara; Reyes-Garcés, Nathaly; Poole, Justen J; Gómez-Ríos, Germán Augusto; Teixeira, Alexandre; Nicol, Beate; Pawliszyn, Janusz

2018-01-18

In vitro high-throughput non-depletive quantitation of chemicals in biofluids is of growing interest in many areas. Some of the challenges facing researchers include the limited volume of biofluids, rapid and high-throughput sampling requirements, and the lack of reliable methods. Coupled to the above, growing interest in the monitoring of kinetics and dynamics of miniaturized biosystems has spurred the demand for development of novel and revolutionary methodologies for analysis of biofluids. The applicability of solid-phase microextraction (SPME) is investigated as a potential technology to fulfill the aforementioned requirements. As analytes with sufficient diversity in their physicochemical features, nicotine, N,N-Diethyl-meta-toluamide, and diclofenac were selected as test compounds for the study. The objective was to develop methodologies that would allow repeated non-depletive sampling from 96-well plates, using 100 µL of sample. Initially, thin film-SPME was investigated. Results revealed substantial depletion and consequent disruption in the system. Therefore, new ultra-thin coated fibers were developed. The applicability of this device to the described sampling scenario was tested by determining the protein binding of the analytes. Results showed good agreement with rapid equilibrium dialysis. The presented method allows high-throughput analysis using small volumes, enabling fast reliable free and total concentration determinations without disruption of system equilibrium.

Americium(III) capture using phosphonic acid-functionalized silicas with different mesoporous morphologies: adsorption behavior study and mechanism investigation by EXAFS/XPS.

PubMed

Zhang, Wen; He, Xihong; Ye, Gang; Yi, Rong; Chen, Jing

2014-06-17

Efficient capture of highly toxic radionuclides with long half-lives such as Americium-241 is crucial to prevent radionuclides from diffusing into the biosphere. To reach this purpose, three different types of mesoporous silicas functionalized with phosphonic acid ligands (SBA-POH, MCM-POH, and BPMO-POH) were synthesized via a facile procedure. The structure, surface chemistry, and micromorphology of the materials were fully characterized by (31)P/(13)C/(29)Si MAS NMR, XPS, and XRD analysis. Efficient adsorption of Am(III) was realized with a fast rate to reach equilibrium (within 10 min). Influences including structural parameters and functionalization degree on the adsorption behavior were investigated. Slope analysis of the equilibrium data suggested that the coordination with Am(III) involved the exchange of three protons. Moreover, extended X-ray absorption fine structure (EXAFS) analysis, in combination with XPS survey, was employed for an in-depth probe into the binding mechanism by using Eu(III) as a simulant due to its similar coordination behavior and benign property. The results showed three phosphonic acid ligands were coordinated to Eu(III) in bidentate fashion, and Eu(P(O)O)3(H2O) species were formed with the Eu-O coordination number of 7. These phosphonic acid-functionalized mesoporous silicas should be promising for the treatment of Am-containing radioactive liquid waste.

Spectroscopic studies on the interaction of sodium benzoate, a food preservative, with calf thymus DNA.

PubMed

Zhang, Guowen; Ma, Yadi

2013-11-01

The interaction between sodium benzoate (SB) and calf thymus DNA in simulated physiological buffer (pH 7.4) using acridine orange (AO) dye as a fluorescence probe, was investigated by UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy along with DNA melting studies and viscosity measurements. An expanded UV-Vis spectral data matrix was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The equilibrium concentration profiles and the pure spectra for SB, DNA and DNA-SB complex from the high overlapping composite response were simultaneously obtained. The results indicated that SB could bind to DNA, and hydrophobic interactions and hydrogen bonds played a vital role in the binding process. Moreover, SB was able to quench the fluorescence of DNA-AO complex through a static procedure. The quenching observed was indicative of an intercalative mode of interaction between SB and DNA, which was supported by melting studies, viscosity measurements and CD analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.

PubMed

Treves, S; Feriotto, G; Moccagatta, L; Gambari, R; Zorzato, F

2000-12-15

Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-11-11

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed Central

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-01-01

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

Tc-99m galactosyl-neoglycoalbumin: in vitro characterization of receptor-mediated binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.

1984-07-01

Hepatic binding protein (HBP) is a membrane receptor that binds and transports plasma glycoproteins from hepatic blood to hepatocellular lysosomes. A characterization is made of the in vitro binding of Tc-99m galactosyl-neoglycoalbumin (Tc-NGA), a synthetic HBP ligand, to liver membrane. Structural modifications of NGA resulted in the alteration of the equilibrium constant, KA, and the forward-binding rate constant, kb. Binding was second-order; the relative amount of membrane-bound NGA depended on the initial concentrations of ligand and membrane. Membrane displacement studies, using carrier ligands in contrast to previously bound Tc-NGA or I-NGA, correlated with the binding characteristics of a native HBPmore » ligand, asialo-orosomucoid. Computer simulation was used to study the detectability of the changes in HBP concentration at different values of kb. The simulations indicated that radiopharmacokinetic sensitivity to alterations in (HBP) should be possible using a neoglycoalbumin preparation with a carbohydrate density within the range of 15 to 25 galactose units per albumin molecule.« less

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes

PubMed Central

Randazzo, Paul A; Jian, Xiaoying; Chen, Pei-Wen; Zhai, Peng; Soubias, Olivier; Northup, John K

2014-01-01

The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5–13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors. PMID:25332840

Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2017-10-01

The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

Characterization of the Physicochemical Properties of β-Cyclodextrin-Divinyl Sulfone Polymer Carrier-Bile Acid Systems.

PubMed

Mohamed, Mohamed H; Wang, Chen; Peru, Kerry M; Headley, John V; Wilson, Lee D

2017-08-07

Herein, we report on the systematic design and characterization of cross-linked polymer carriers containing β-cyclodextrin (β-CD) and divinyl sulfone (DVS). The polymer carriers were prepared at variable feed ratios (β-CD-DVS; 1:1, 1:2, 1:3, and 1:6) and characterized using spectroscopy (IR, 1 H solution NMR, and 13 C CP-MAS solids NMR spectroscopy), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and a dye decolorization method using phenolphthalein. Uptake studies were carried out at pH 9.00 for the polymer carriers using single component bile acids (cholic acid, deoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid). Equilibrium uptake results were evaluated by the Langmuir isotherm model where variable equilibrium parameters were related to the relative apolar character of the bile acid. The Langmuir model yields a carrier/bile acid binding affinity of ∼10 3 M -1 where the lipophilic inclusion sites of the polymer play a prominent role, while the DVS linker framework sites have a lower adsorption affinity, in accordance with the greater hydrophilic character of such sites.

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

PubMed

Chétrite, G; Cassoly, R

1985-10-05

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Shift in the equilibrium between on and off states of the allosteric switch in Ras-GppNHp affected by small molecules and bulk solvent composition.

PubMed

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-07

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

Origin of the size-dependence of the equilibrium van der Waals binding between nanostructures

NASA Astrophysics Data System (ADS)

Tao, Jianmin; Perdew, John P.; Tang, Hong; Shahi, Chandra

2018-02-01

Nanostructures can be bound together at equilibrium by the van der Waals (vdW) effect, a small but ubiquitous many-body attraction that presents challenges to density functional theory. How does the binding energy depend upon the size or number of atoms in one of a pair of identical nanostructures? To answer this question, we treat each nanostructure as a whole object, not as a collection of atoms. Our calculations start from an accurate static dipole polarizability for each considered nanostructure, and an accurate equilibrium center-to-center distance for the pair (the latter from experiment or from the vdW-DF-cx functional). We consider the competition in each term -C2k/d2k (k = 3, 4, 5) of the long-range vdW series for the interaction energy, between the size dependence of the vdW coefficient C2k and that of the 2kth power of the center-to-center distance d. The damping of these vdW terms can be negligible, but in any case, it does not affect the size dependence for a given term in the absence of non-vdW binding. To our surprise, the vdW energy can be size-independent for quasi-spherical nanoclusters bound to one another by vdW interaction, even with strong nonadditivity of the vdW coefficient, as demonstrated for fullerenes. We also show that, for low-dimensional systems, the vdW interaction yields the strongest size-dependence, in stark contrast to that of fullerenes. We illustrate this with parallel planar polycyclic aromatic hydrocarbons. The size dependences of other morphologies or bonding types lie between, as shown by sodium clusters.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

PubMed

Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

2000-12-01

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

PubMed Central

Scott, D J; Ferguson, A L; Gallegos, M T; Pitt, M; Buck, M; Hoggett, J G

2000-01-01

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed. PMID:11085949

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

Origin of the size-dependence of the equilibrium van der Waals binding between nanostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jianmin

Nanostructures can be bound together at equilibrium by the van der Waals (vdW) effect, a small but ubiquitous many-body attraction that presents challenges to density functional theory. How does the binding energy depend upon the size or number of atoms in one of a pair of identical nanostructures? To answer this question, we treat each nanostructure properly as a whole object, not as a collection of atoms. Our calculations start from an accurate static dipole polarizability for each considered nanostructure, and an accurate equilibrium center-to-center distance for the pair (the latter from experiment, or from the vdW-DF-cx functional). We consider the competition in each termmore » $$-C_{2k}/d^{2k}$$ ($k=3, 4, 5$) of the long-range vdW series for the interaction energy, between the size dependence of the vdW coefficient $$C_{2k}$$ and that of the $2k$-th power of the center-to-center distance $d$. The damping of these vdW terms can be negligible, but in any case it does not affect the size dependence for a given term in the absence of non-vdW binding. To our surprise, the vdW energy can be size-independent for quasi-spherical nanoclusters bound to one another by vdW interaction, even with strong nonadditivity of the vdW coefficient, as demonstrated for fullerenes. We also show that, for low-dimensional systems, the vdW interaction yields the strongest size-dependence, in stark contrast to that of fullerenes. We illustrate this with parallel planar polycyclic aromatic hydrocarbons. Other cases are between, as shown by sodium clusters.« less

Origin of the size-dependence of the equilibrium van der Waals binding between nanostructures

DOE PAGES

Tao, Jianmin

2018-02-21

Nanostructures can be bound together at equilibrium by the van der Waals (vdW) effect, a small but ubiquitous many-body attraction that presents challenges to density functional theory. How does the binding energy depend upon the size or number of atoms in one of a pair of identical nanostructures? To answer this question, we treat each nanostructure properly as a whole object, not as a collection of atoms. Our calculations start from an accurate static dipole polarizability for each considered nanostructure, and an accurate equilibrium center-to-center distance for the pair (the latter from experiment, or from the vdW-DF-cx functional). We consider the competition in each termmore » $$-C_{2k}/d^{2k}$$ ($k=3, 4, 5$) of the long-range vdW series for the interaction energy, between the size dependence of the vdW coefficient $$C_{2k}$$ and that of the $2k$-th power of the center-to-center distance $d$. The damping of these vdW terms can be negligible, but in any case it does not affect the size dependence for a given term in the absence of non-vdW binding. To our surprise, the vdW energy can be size-independent for quasi-spherical nanoclusters bound to one another by vdW interaction, even with strong nonadditivity of the vdW coefficient, as demonstrated for fullerenes. We also show that, for low-dimensional systems, the vdW interaction yields the strongest size-dependence, in stark contrast to that of fullerenes. We illustrate this with parallel planar polycyclic aromatic hydrocarbons. Other cases are between, as shown by sodium clusters.« less

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-31

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) canmore » selectively shift the equilibrium to the 'on' state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the 'ordered off' state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-{beta}. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.« less

Mechanism of increased clearance of glycated albumin by proximal tubule cells

PubMed Central

Wagner, Mark C.; Myslinski, Jered; Pratap, Shiv; Flores, Brittany; Rhodes, George; Campos-Bilderback, Silvia B.; Sandoval, Ruben M.; Kumar, Sudhanshu; Patel, Monika; Ashish

2016-01-01

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins. PMID:26887834

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

PubMed Central

Almaqwashi, Ali A.; Andersson, Johanna; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

2016-01-01

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2]4+ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics. PMID:27028636

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

PubMed

Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

2012-03-01

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

PubMed

Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.

Dynamic acid/base equilibrium in single component switchable ionic liquids and consequences on viscosity

DOE PAGES

Cantu, David C.; Lee, Juntaek; Lee, Mal -Soon; ...

2016-03-28

The deployment of transformational non-aqueous CO 2-capture solvent systems is encumbered by high viscosity even at intermediate uptakes. Using single-molecule CO 2 binding organic liquids as a prototypical example, we identify the key molecular features controlling bulk liquid viscosity and CO 2 uptake kinetics. Fast uptake kinetics arise from close proximity of the alcohol and amine sites that are involved in CO 2 binding. This process results in the concerted formation of a Zwitterion containing both an alkylcarbonate and a protonated amine. The hydrogen bonding between the two functional groups ultimately determines the solution viscosity. Based on molecular simulation, thismore » work reveals options to significantly reduce viscosity with molecular modifications that shift the proton transfer equilibrium towards a neutral acid/amine species as opposed to the ubiquitously accepted Zwitterionic state. Lastly, the molecular design concepts proposed here, for the alkyl-carbonate systems, are readily extensible to other CO 2 capture technologies, such as the carbamate- or imidazole-based solvent chemistries.« less

Thermodynamics of the interaction of sweeteners and lactisole with fullerenols as an artificial sweet taste receptor model.

PubMed

Chen, Zhong-Xiu; Wu, Wen; Zhang, Wei-Bin; Deng, Shao-Ping

2011-09-01

The thermodynamics of the mimetic interaction of lactisole and sweeteners with fullerenols as a synthetic sweet receptor model was elucidated by Isothermal Titration Calorimetry (ITC) technique. The presence of lactisole resulted in great differences in thermodynamics of the sweeteners binding with fullerenols in which lactisole led to much more entropy contribution to the free energy compared with the interaction of sweeteners with fullerenols. Two interaction equilibrium states were found in ITC titration profiles and competitive binding of lactisole and sweeteners with fullerenols was disclosed. Our results indicated that the larger value of the ratio of two equilibrium constant K1/K2, the more effectively lactisole inhibited the sweetness of the sweetener. The combined results of sensory evaluation and ITC thermodynamics revealed that introducing a synthetic receptor model to interact with the sweeteners and inhibitors helps to understand the inhibition mechanism and the thermodynamic basis for the initiation of sweetness inhibition. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biosorption of metal ions from aqueous solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Jiaping; Yiacoumi, Sotira

1997-01-01

Copper biosorption from aqueous solutions by calcium alginate is reported in this paper. The experimental section includes potentiometric titrations of biosorbents, batch equilibrium and kinetic studies of copper biosorption, as well as fixed-bed biosorption experiments. The potentiometric titration results show that the surface charge increases with decreasing pH. The biosorption of copper strongly depends on solution pH; the metal ion binding increases from 0 to 90 percent in pH ranging from 1.5 to 5.0. In addition, a decrease in ionic strength results in an increase of copper ion removal. Kinetic studies indicate that mass transfer plays an important role inmore » the biosorption rate. Furthermore, a fixed-bed biosorption experiment shows that calcium alginate has a significant capacity for copper ion removal. The two-pK Basic Stem model successfully represents the surface charge and equilibrium biosorption experimental data. The calculation results demonstrate that the copper removal may result from the binding of free copper and its hydroxide with surface functional groups of the biosorbents.« less

Studies on molecular structure, vibrational spectra and molecular docking analysis of 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate.

PubMed

Suresh, D M; Amalanathan, M; Joe, I Hubert; Jothy, V Bena; Diao, Yun-Peng

2014-09-15

The molecular structure, vibrational analysis and molecular docking analysis of the 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate (MDDNAB) molecule have been carried out using FT-IR and FT-Raman spectroscopic techniques and DFT method. The equilibrium geometry, harmonic vibrational wave numbers, various bonding features have been computed using density functional method. The calculated molecular geometry has been compared with experimental data. The detailed interpretation of the vibrational spectra has been carried out by using VEDA program. The hyper-conjugative interactions and charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The simulated FT-IR and FT-Raman spectra satisfactorily coincide with the experimental spectra. The PES and charge analysis have been made. The molecular docking was done to identify the binding energy and the Hydrogen bonding with the cancer protein molecule. Copyright © 2014 Elsevier B.V. All rights reserved.

Hemoglobin Rahere, a human hemoglobin variant with amino acid substitution at the 2,3-diphosphoglycerate binding site. Functional consequences of the alteration and effects of bezafibrate on the oxygen bindings.

PubMed

Sugihara, J; Imamura, T; Nagafuchi, S; Bonaventura, J; Bonaventura, C; Cashon, R

1985-09-01

We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A.

Hemoglobin Rahere, a human hemoglobin variant with amino acid substitution at the 2,3-diphosphoglycerate binding site. Functional consequences of the alteration and effects of bezafibrate on the oxygen bindings.

PubMed Central

Sugihara, J; Imamura, T; Nagafuchi, S; Bonaventura, J; Bonaventura, C; Cashon, R

1985-01-01

We encountered an abnormal hemoglobin (Rahere), with a threonine residue replacing the beta 82 (EF6) lysine residue at the binding site of 2,3-diphosphoglycerate, which was responsible for overt erythrocytosis in two individuals of a Japanese family. Hemoglobin Rahere shows a lower oxygen affinity on the binding of 2,3-diphosphoglycerate or chloride ions than hemoglobin A. Although a decrease in the positive charge density at the binding sites of 2,3-diphosphoglycerate in hemoglobin Rahere apparently shifts the allosteric equilibrium toward the low affinity state, it greatly diminishes the cofactor effects by anions. The oxygen affinity of the patient's erythrocytes is substantially lowered by the presence of bezafibrate, which combines with sites different from those of 2,3-diphosphoglycerate in either hemoglobin Rahere or hemoglobin A. PMID:3930571

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

Binding of Thyrotropin-Releasing Hormone to Plasma Membranes of Bovine Anterior Pituitary Gland

PubMed Central

Labrie, Fernand; Barden, Nicholas; Poirier, Guy; De Lean, Andre

1972-01-01

An assay for the binding of [3H]thyrotropin-releasing hormone ([3H]TRH) is described. Plasma membranes isolated from bovine anterior pituitary gland bind about 600 femtomoles of this hormone per mg of protein, as compared to 15 femtomoles per mg of protein in the total adenohypophyseal homogenate (40-fold purification). The equilibrium constant of membrane receptor-[3H]TRH binding at 0°C is 4.3 × 107 L·M-1, or a half-maximal binding of this hormone at 23 nM. The binding is time-dependent; addition of unlabeled hormone induces dissociation of the receptor-[3H]TRH complex with a half-life of 14 min. The binding of TRH is not altered by 10 μM melanocyte-stimulating hormone-release inhibiting hormone, lysine-vasopressin, adrenocorticotropin, growth hormone, prolactin, luteinizing hormone, insulin, glucagon, L-thyroxine, or L-triiodothyronine. K+ and Mg++ increase formation of the receptor-TRH complex at optimal concentrations of 5-25 mM and 0.5-2.5 mM, respectively, with inhibition at higher concentrations. Ca++ inhibits binding of TRH at all concentrations tested. PMID:4621548

Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

PubMed Central

Huang, Tao; Long, Mian; Huo, Bo

2010-01-01

Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

Immunomagnetic separation of human myeloperoxidase using an antibody-mimicking peptide identified by phage display.

PubMed

Yun, Soi; Ryu, Hyunmin; Lee, E K

2017-09-10

Phage display biopanning is a powerful in vitro selection process for screening and identifying peptides that bind to a target protein of interest. With the aim of replacing antibodies in immuno-diagnostic applications, we identified peptides whose binding characteristics mimicked those of anti-human myeloperoxidase (hMPO), a biomarker for acute cardiac diseases. Based on ELISA results from four phage clones, we selected and chemically synthesized a 12-mer peptide (SYIEPPERHRHR). Quartz crystal microbalance and surface plasmon resonance analyses revealed that the molar binding equilibrium ratio of the synthesized peptide was 0.023, approximately 43-fold lower than that of the anti-hMPO antibody. The dissociation constant (K d ) was 57nM, which was comparable to that of the native antibody (83nM). Next, we biotinylated the peptide at its N-terminus and attached the biotinylated peptide to the surface of streptavidin-coated magnetic particles to assess its ability to selectively capture hMPO. The binding equilibrium data were similar to the previous analyses; specifically, around 0.021mol peptide bound to 1mol of hMPO. Antigen capture was found to be selective and to be relatively little influenced by the presence of human serum albumin (HSA), an abundant constituent of serum. Our work demonstrates the potential of immunomagnetic isolation to achieve selective capture of a low-concentration antigen from complex solutions such as serum. Copyright © 2016 Elsevier B.V. All rights reserved.

THE EFFECTS OF TYPE II BINDING ON METABOLIC STABILITY AND BINDING AFFINITY IN CYTOCHROME P450 CYP3A4

PubMed Central

Peng, Chi-Chi; Pearson, Josh T.; Rock, Dan A.; Joswig-Jones, Carolyn A.; Jones, Jeffrey P.

2010-01-01

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding. PMID:20346909

Thermodynamics of proton transport coupled ATP synthesis.

PubMed

Turina, Paola; Petersen, Jan; Gräber, Peter

2016-06-01

The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. Copyright © 2016 Elsevier B.V. All rights reserved.

Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

PubMed

Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J

2018-05-25

Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Interaction of Sodium Hyaluronate with a Biocompatible Cationic Surfactant from Lysine: A Binding Study.

PubMed

Bračič, Matej; Hansson, Per; Pérez, Lourdes; Zemljič, Lidija F; Kogej, Ksenija

2015-11-10

Mixtures of natural and biodegradable surfactants and ionic polysaccharides have attracted considerable research interest in recent years because they prosper as antimicrobial materials for medical applications. In the present work, interactions between the lysine-derived biocompatible cationic surfactant N(ε)-myristoyl-lysine methyl ester, abbreviated as MKM, and the sodium salt of hyaluronic acid (NaHA) are investigated in aqueous media by potentiometric titrations using the surfactant-sensitive electrode and pyrene-based fluorescence spectroscopy. The critical micelle concentration in pure surfactant solutions and the critical association concentration in the presence of NaHA are determined based on their dependence on the added electrolyte (NaCl) concentration. The equilibrium between the protonated (charged) and deprotonated (neutral) forms of MKM is proposed to explain the anomalous binding isotherms observed in the presence of the polyelectrolyte. The explanation is supported by theoretical model calculations of the mixed-micelle equilibrium and the competitive binding of the two MKM forms to the surface of the electrode membrane. It is suggested that the presence of even small amounts of the deprotonated form can strongly influence the measured electrode response. Such ionic-nonionic surfactant mixtures are a special case of mixed surfactant systems where the amount of the nonionic component cannot be varied independently as was the case for some of the earlier studies.

Effects of methyl substitution on DNA binding enthalpies of enantiopure Ru(phenanthroline)2dipyridophenazine2+ complexes.

PubMed

Mårtensson, Anna K F; Lincoln, Per

2018-04-25

Isothermal titration calorimetry (ITC) has been utilized to investigate the effect of methyl substituents on the intercalating dppz ligand of the enantiomers of the parent complex Ru(phen)2dppz2+ (phen = 1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) on DNA binding thermodynamics. The methylated complexes (10-methyl-dppz and 11,12-dimethyl-dppz) have large, concentration-dependent, positive heats of dilution, and a strong endothermic background is also apparent in the ITC-profiles from titration of methylated complexes into poly(dAdT)2, which make direct comparison between complexes difficult. By augmenting a simple cooperative binding model with one equilibrium for complex self-aggregation in solution and one equilibrium for complex aggregation on saturated DNA, it was possible to find an excellent global fit to the experimental data with DNA affinity parameters restricted to be equal for all Δ-enantiomers as well as for all Λ-enantiomers. In general, enthalpic differences, compared to the unsubstituted complex, were small and less than 4 kJ mol-1, except for the heat of intercalation of Δ-10-methyl-dppz (-11,6 kJ mol-1) and Λ-11,12-dimethyl-dppz (+4.3 kJ mol-1).

Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I.

PubMed Central

Hoggett, J G; Kellett, G L

1995-01-01

Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state. Images Figure 1 PMID:7832753

Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwarz, S.; Ellen, R.P.; Grove, D.A.

1987-10-01

There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HAmore » (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.« less

Isocyanides inhibit human heme oxygenases at the verdoheme stage.

PubMed

Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

2009-09-22

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

PubMed Central

Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

2010-01-01

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Divers models of divalent cation interaction to calcium-binding proteins: techniques and anthology.

PubMed

Cox, Jos A

2013-01-01

Intracellular Ca(2+)-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca(2+). Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg(2+) concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca(2+) indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-type CaBPs were published. With the advent of recombinant CaBPs it became possible to determine these properties on previously inaccessible proteins. Here I report on studies by our group carried out in the last decade on eight families of recombinant CaBPs, their mutants, or truncated domains. Moreover this chapter deals with the currently used methods for quantifying the binding of Ca(2+) and Mg(2+) to CaBPs.

Decorin is a Zn(2+) Metalloprotein

NASA Technical Reports Server (NTRS)

Yang, Vivian W.-C.; LaBrenz, Steven R.; Rosenberg, Lawrence C.; McQuillan, David; Hoeoek, Magnus

1998-01-01

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

PubMed

Chiara, David C; Trinidad, Jonathan C; Wang, Dong; Ziebell, Michael R; Sullivan, Deirdre; Cohen, Jonathan B

2003-01-21

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

Evaluation of water binding, seed coat permeability and germination characteristics of wheat seeds equilibrated at different relative humidities.

PubMed

Chatterjee, Nabamita; Nagarajan, Shantha

2006-08-01

The relative binding of seed water and seed coat membrane stability were measured in two contrasting wheat (Triticum aestivum L) varieties, HDR 77 (drought-tolerant) and HD 2009 (susceptible) using seed water sorption isotherms, electrical conductivity (EC) of leachates and desorption-absorption isotherms. Analysis of sorption isotherm at 25 degrees C showed that the seeds of HDR 77 had significantly higher number of strong binding sites, with correspondingly greater amount of seed water as strongly bound water, as compared to HD 2009. Total number of binding sites was also higher in HDR 77 than HD 2009, which explained the better desiccation tolerance and higher capacity to bind water in seeds of HDR 77. EC of seed leachate in both varieties did not change with respect to change in equilibrium relative humidity (RII), indicating the general seed coat membrane stability of wheat seeds. However, absolute conductivity values were higher for HD 2009. showing its relatively porous seed coat membrane. Significantly lower area enclosed by the desorption-absorption isotherm loop in HDR 77, as compared to HD 2009 also indicated the greater membrane integrity of HDR 77. Germination and seedling vigour of HD 2009 were reduced when equilibrated over very low and very high RH. In contrast, germination and vigour in HDR 77 were maintained high, except at very high RH, indicating again its desiccation tolerance. Thus, the study demonstrated the relative drought tolerance of HDR 77, on the basis of seed water-binding characteristics and seed membrane stability. Seed membrane stability as measured by seed leachate conductivity or as area under dehydration-rehydration loop may be used as a preliminary screening test for drought tolerance in wheat.

Equilibrium modeling of 5-HT(2A) receptors with [18F]deuteroaltanserin and PET: feasibility of a constant infusion paradigm.

PubMed

van Dyck, C H; Soares, J C; Tan, P Z; Staley, J K; Baldwin, R M; Amici, L A; Fu, X; Garg, P K; Seibyl, J P; Charney, D S; Innis, R B

2000-11-01

[(18)F]Altanserin has emerged as a promising positron emission tomography (PET) ligand for serotonin-2A (5-HT(2A)) receptors. The deuterium substitution of both of the 2'-hydrogens of altanserin ([(18)F]deuteroaltanserin) yields a metabolically more stable radiotracer with higher ratios of parent tracer to radiometabolites and increased specific brain uptake than [(18)F]altanserin. The slower metabolism of the deuterated analog might preclude the possibility of achieving stable plasma and brain activities with a bolus plus constant infusion within a reasonable time frame for an (18)F-labeled tracer (T(1/2) 110 min). Thus, the purpose of this study was to test the feasibility in human subjects of a constant infusion paradigm for equilibrium modeling of [(18)F]deuteroaltanserin with PET. Seven healthy male subjects were injected with [(18)F]deuteroaltanserin as a bolus plus constant infusion lasting 10 h postinjection. PET acquisitions and venous blood sampling were performed throughout the infusion period. Linear regression analysis revealed that time-activity curves for both specific brain uptake and plasma [(18)F]deuteroaltanserin concentration stabilized after about 5 h. This permitted equilibrium modeling and estimation of V(')(3) (ratio of specific uptake to total plasma parent concentration) and the binding potential V(3) (ratio of specific uptake to free plasma parent concentration). Cortical/cerebellar ratios were increased by 26% relative to those we previously observed with [(18)F]altanserin using similar methodology in a somewhat older subject sample. These results demonstrate feasibility of equilibrium imaging with [(18)F]deuteroaltanserin and suggest that it may be superior to [(18)F]altanserin as a PET radioligand.

Conformational flexibility of arginine-82 as source for the heterogeneous and pH-dependent kinetics of the primary proton transfer step in the bacteriorhodopsin photocycle: An electrostatic model

NASA Astrophysics Data System (ADS)

Scharnagl, Christina; Fischer, Sighart F.

1996-11-01

We use equilibrium thermodynamic concepts to relate protein conformational and protonation substates and their pH-dependent population to kinetic schemes for the rise of the M intermediate in the photocycle of bacteriorhodopsin. Conformational flexibility of arginine R82 is described by a two-state model. The analysis accounts for the electrostatic coupling between its orientation and hydrogen ion titration and presents a structural basis for the linkage between the protonation states of the primary proton acceptor, aspartate D85, and the extracellular release group, glutamate E204. We find that the charge state of D85 is a significant determinant for the orientation of R82. The molecular model predicts the following: the primary proton transfer to D85 can be described by a kinetic scheme with two heterogeneous substates. They control the event with different activation parameters due to the reorientation of R82 away from the chromophore binding site. Their population depends on the external pH and the proton exchange equilibrium between the membrane buried residues and the bulk aqueous solvent. Proton transfer in the physiologic pH range is strongly activated and followed by the reorientation of R82 which shifts the equilibrium toward complete transfer. In the alkaline pH region a different mechanism operates, which involves the increased population of a substate with already reoriented R82 as a consequence of the deprotonation of E204, leading to accelerated proton transfer. Assuming full proton exchange equilibrium with the bulk water on the millisecond time scale leads to an increased population of substates which are non-productive for proton transfer.

Lead sorption by waste biomass of hazelnut and almond shell.

PubMed

Pehlivan, Erol; Altun, Türkan; Cetin, Serpil; Iqbal Bhanger, M

2009-08-15

The potential to remove Pb(2+) ion from aqueous solutions using the shells of hazelnut (HNS) (Corylus avellana) and almond (AS) (Prunus dulcis) through biosorption was investigated in batch experiments. The main parameters influencing Pb(2+) ion sorption on HNS and AS were: initial metal ion concentration, amount of adsorbent, contact time and pH value of solution. The influences of initial Pb(2+) ion concentration (0.1-1.0mM), pH (2-9), contact time (10-240 min) and adsorbent amount (0.1-1.0 g) have been investigated. Equilibrium isotherms have been measured and modelled. Adsorption of Pb(2+) ions was in all cases pH-dependent showing a maximum at equilibrium pH values between 6.0 and 7.0, depending on the biomaterial, that corresponded to equilibrium pH values of 6.0 for HNS and 7.0 for AS. The equilibrium sorption capacities of HNS and AS were 28.18 and 8.08 mg/g for lead, respectively after equilibrium time of 2h. The adsorption data fit well with the Langmuir isotherm model and the experimental result inferred that adsorption, chelation and ion exchange are major adsorption mechanisms for binding Pb(2+) ion to the sorbents.

Anomalous versus Slowed-Down Brownian Diffusion in the Ligand-Binding Equilibrium

PubMed Central

Soula, Hédi; Caré, Bertrand; Beslon, Guillaume; Berry, Hugues

2013-01-01

Measurements of protein motion in living cells and membranes consistently report transient anomalous diffusion (subdiffusion) that converges back to a Brownian motion with reduced diffusion coefficient at long times after the anomalous diffusion regime. Therefore, slowed-down Brownian motion could be considered the macroscopic limit of transient anomalous diffusion. On the other hand, membranes are also heterogeneous media in which Brownian motion may be locally slowed down due to variations in lipid composition. Here, we investigate whether both situations lead to a similar behavior for the reversible ligand-binding reaction in two dimensions. We compare the (long-time) equilibrium properties obtained with transient anomalous diffusion due to obstacle hindrance or power-law-distributed residence times (continuous-time random walks) to those obtained with space-dependent slowed-down Brownian motion. Using theoretical arguments and Monte Carlo simulations, we show that these three scenarios have distinctive effects on the apparent affinity of the reaction. Whereas continuous-time random walks decrease the apparent affinity of the reaction, locally slowed-down Brownian motion and local hindrance by obstacles both improve it. However, only in the case of slowed-down Brownian motion is the affinity maximal when the slowdown is restricted to a subregion of the available space. Hence, even at long times (equilibrium), these processes are different and exhibit irreconcilable behaviors when the area fraction of reduced mobility changes. PMID:24209851

Structural basis of oligosaccharide processing by glycosaminoglycan sulfotransferases.

PubMed

Gesteira, Tarsis F; Coulson-Thomas, Vivien J

2018-06-06

Heparan sulfate (HS) is a sulfated polysaccharide that plays a key role in morphogenesis, physiology and pathogenesis. The biosynthesis of HS takes place in the Golgi apparatus by a group of enzymes that polymerize, epimerize and sulfate the sugar chain. This biosynthetic process introduces varying degrees of sulfate substitution, which are tightly regulated and directly dictate binding specificity to different cytokines, morphogens and growth factors. Here we report the use of molecular dynamics simulations to investigate the dynamics of substrate recognition of two glycosaminoglycan (GAG) sulfotransferases, N-deacetylase-N-sulfotransferase and 2-O-sulfotransferase to the HS chain during the biosynthetic process. We performed multiple simulations of the binding of the sulfotransferase domains to both the HS oligosaccharide substrate and sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPs). Analysis of extended simulations provide detailed and useful insights into the atomic interactions that are at work during oligosaccharide processing. The Fast Information Matching method was used to detect the enzyme global dynamics and to predict the pairwise contact of residues responsible for GAG-enzyme binding and unbinding. The correlation between HS displacement and the location of the modified GAG chain were calculated, indicating a possible route for HS and heparin during sulfotransferase processing. Our data also show sulfotransferases contain a conserved interspaced positively charged amino acid residues that form a patch which controls the protein-GAG binding equilibrium. Together, our findings provide further understanding on the fine-tuned complex mechanism of GAG biosynthesis. Our findings can also be extrapolated to other systems for calculating rates of protein-GAG binding.

Carbon-Binding Designer Proteins that Discriminate between sp2- and sp3-Hybridized Carbon Surfaces

PubMed Central

Coyle, Brandon L.; Rolandi, Marco; Baneyx, François

2013-01-01

Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. Here, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of E. coli Thioredoxin 1 (TrxA). We develop purification schemes to recover the resulting TrxA derivatives in a soluble form and conduct a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbonaceous surfaces. Although equilibrium quartz crystal microbalance measurements show that TrxA∷Car9 and TrxA∷Car15 have similar affinity for sp2-hybridized graphitic carbon (Kd = 50 and 90 nM, respectively), only the latter protein is capable of dispersing carbon nanotubes. Further investigation by surface plasmon resonance and atomic force microscopy reveals that TrxA∷Car15 interacts with sp2-bonded carbon through a combination of hydrophobic and π-π interactions but that TrxA∷Car9 exhibits a cooperative mode of binding which relies on a combination of electrostatics and weaker π-stacking. Consequently, we find that TrxA∷Car9 binds equally well to sp2- and sp3-bonded (diamond-like) carbon particles, while TrxA∷Car15 is capable of discriminating between the two carbon allotropes. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications. PMID:23510486

Simulating Molecular Mechanisms of the MDM2-Mediated Regulatory Interactions: A Conformational Selection Model of the MDM2 Lid Dynamics

PubMed Central

Verkhivker, Gennady M.

2012-01-01

Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between “closed” and “semi-closed” lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of “semi-closed” conformations. The dominant “semi-closed” lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics. PMID:22815859

Binding affinities of cationic dyes in the presence of activated charcoal and anionic surfactant in the premicellar region

NASA Astrophysics Data System (ADS)

Ali, Farman; Ibrahim, Muhammad; Khan, Fawad; Bibi, Iram; Shah, Syed W. H.

2018-03-01

Binding preferences of cationic dyes malachite green and methylene blue in a mixed charcoal-sodium dodecyl sulfate system have been investigated using UV-visible absorption spectroscopy. The dye adsorption shows surfactant-dependent patterns, indicating diverse modes of interactions. At low surfactant concentration, a direct binding to charcoal is preferred. Comparatively greater quantities of surfactant lead to attachment of dye-surfactant complex to charcoal through hydrophobic interactions. A simple model was employed for determination of equilibrium constant K eq and concentration of dye-surfactant ion pair N DS for both dyes. The values of binding parameters revealed that malachite green was directly adsorbed onto charcoal, whereas methylene blue was bound through surfactant monomers. The model is valid for low surfactant concentrations in the premicellar region. These findings have significance for material and environmental sciences.

Electrochemical and spectroscopic studies of the interaction of proflavine with DNA.

PubMed

Aslanoglu, Mehmet

2006-03-01

The interaction of proflavine with herring sperm DNA has been investigated by cyclic voltammetry and UV-Vis spectroscopy as well as viscosity measurements. Shifts in the peak potentials in cyclic voltammetry, spectral changes in UV absorption titration, an increase in viscosity of DNA and the results of the effect of ionic strength on the binding constant strongly support the intercalation of proflavine into the DNA double helix. The binding constant for the interaction between proflavine and DNA was K = 2.32 (+/- 0.41) x 10(4) M(-1) and the binding site size was 2.07 (+/- 0.1) base pairs, estimated in voltammetric measurements. The value of the binding site size was determined to be closer to that expected for a planar intercalating agent. The standard Gibbs free-energy change is ca. -24.90 kJ/mol at 25 degrees C, indicating the spontaneity of the binding interaction. The binding constant determined by UV absorption measurements was K = 2.20 (+/- 0.48) x 10(4) M(-1), which is very close to the value determined by cyclic voltammetry assuming that the binding equilibrium is static.

Identification of gold sensing peptide by integrative proteomics and a bacterial two-component system

NASA Astrophysics Data System (ADS)

Ng, I.-Son; Yu, You-Jin; Yi, Ying-Chen; Tan, Shih-I.; Huang, Bo-Chuan; Han, Yin-Lung

2017-12-01

The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel gold binding peptide. Finally, the heterologous over-expression of gold binding peptide by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.

Equilibrium Gel Filtration Chromatography for the Measurement of Protein-Ligand Binding in the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Craig, Douglas B.

2005-01-01

A laboratory exercise used in the senior biochemistry course at the University of Winnipeg for three years is discussed. It combines liquid chromatography and absorbance spectroscopy and also allows the students to produce a quantitative result within a single three-hour period.

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

PubMed

Fukada, H; Sturtevant, J M; Quiocho, F A

1983-11-10

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

PubMed Central

Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

2013-01-01

The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron–transfer reactions. PMID:23909369

Kinetic, Thermodynamic, and Structural Insight into the Mechanism of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wubben, Thomas J.; Mesecar, Andrew D.; UIC)

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine (PhP) to form dephosphocoenzyme A. This reaction sits at the branch point between the de novo pathway and the salvage pathway, and has been shown to be a rate-limiting step in the biosynthesis of CoA. Importantly, bacterial and mammalian PPATs share little sequence homology, making the enzyme a potential target for antibiotic development. A series of steady-state kinetic, product inhibition, and direct binding studies with Mycobacterium tuberculosis PPAT (MtPPAT) was conducted and suggests that the enzyme utilizesmore » a nonrapid-equilibrium random bi-bi mechanism. The kinetic response of MtPPAT to the binding of ATP was observed to be sigmoidal under fixed PhP concentrations, but substrate inhibition was observed at high PhP concentrations under subsaturating ATP concentrations, suggesting a preferred pathway to ternary complex formation. Negative cooperativity in the kinetic response of MtPPAT to PhP binding was observed under certain conditions and confirmed thermodynamically by isothermal titration calorimetry, suggesting the formation of an asymmetric quaternary structure during sequential ligation of substrates. Asymmetry in binding was also observed in isothermal titration calorimetry experiments with dephosphocoenzyme A and CoA. X-ray structures of MtPPAT in complex with PhP and the nonhydrolyzable ATP analogue adenosine-5'-[({alpha},{beta})-methyleno]triphosphate were solved to 1.57 {angstrom} and 2.68 {angstrom}, respectively. These crystal structures reveal small conformational changes in enzyme structure upon ligand binding, which may play a role in the nonrapid-equilibrium mechanism. We suggest that the proposed kinetic mechanism and asymmetric character in MtPPAT ligand binding may provide a means of reaction and pathway regulation in addition to that of the previously determined CoA feedback.« less

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

PubMed Central

Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

2015-01-01

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Efficient synthesis of molecularly imprinted polymers with bio-recognition sites for the selective separation of bovine hemoglobin.

PubMed

Zhang, Zulei; Li, Lei

2018-06-01

We developed a facile approach to the construction of bio-recognition sites in silica nanoparticles for efficient separation of bovine hemoglobin based on amino-functionalized silica nanoparticles grafting by 3-aminopropyltriethoxylsilane providing hydrogen bonds with bovine hemoglobin through surface molecularly imprinting technology. The resulting amino-functionalized silica surface molecularly imprinted polymers were characterized using scanning electron microscope, transmission electronic microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Results showed that the as-synthesized imprinted polymers exhibited spherical morphology and favorable thermal stability. The binding adsorption experiments showed that the imprinted polymers can reach equilibrium within 1 h. The Langmuir isotherm and pseudo-second-order kinetic model fitted the adsorption data well. Meanwhile, the imprinted polymers possessed a maximum binding capacity up to 90.3 mg/g and highly selectivity for the recognition of bovine hemoglobin. Moreover, such high binding capacity and selectivity retained after eight cycles, indicating the good stability and reusability of the imprinted polymers. Finally, successful application in the selective recognition of bovine hemoglobin from a real bovine blood sample indicated that the imprinted polymers displayed great potentials in efficient purification and separation of target proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational Equilibrium Thermodynamic and Kinetic Analysis of K-Ras Dimerization through an Effector Binding Surface Suggests Limited Functional Role.

PubMed

Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Hancock, John F; Gorfe, Alemayehu A

2016-08-25

Dimer formation is believed to have a substantial impact on regulating K-Ras function. However, the evidence for dimerization and the molecular details of the process are scant. In this study, we characterize a K-Ras pseudo-C2-symmetric dimerization interface involving the effector interacting β2-strand. We used structure matching and all-atom molecular dynamics (MD) simulations to predict, refine, and investigate the stability of this interface. Our MD simulation suggested that the β2-dimer is potentially stable and remains relatively close to its initial conformation due to the presence of a number of hydrogen bonds, ionic salt bridges, and other favorable interactions. We carried out potential of mean force calculations to determine the relative binding strength of the interface. The results of these calculations indicated that the β2 dimerization interface provides a weak binding free energy in solution and a dissociation constant that is close to 1 mM. Analyses of Brownian dynamics simulations suggested an association rate kon ≈ 10(5)-10(6) M(-1) s(-1). Combining these observations with available literature data, we propose that formation of auto-inhibited β2 K-Ras dimers is possible but its fraction in cells is likely very small under normal physiologic conditions.

Interaction of D2 with H2O amorphous ice studied by temperature-programmed desorption experiments.

PubMed

Amiaud, L; Fillion, J H; Baouche, S; Dulieu, F; Momeni, A; Lemaire, J L

2006-03-07

The gas-surface interaction of molecular hydrogen D2 with a thin film of porous amorphous solid water (ASW) grown at 10 K by slow vapor deposition has been studied by temperature-programmed-desorption (TPD) experiments. Molecular hydrogen diffuses rapidly into the porous network of the ice. The D2 desorption occurring between 10 and 30 K is considered here as a good probe of the effective surface of ASW interacting with the gas. The desorption kinetics have been systematically measured at various coverages. A careful analysis based on the Arrhenius plot method has provided the D2 binding energies as a function of the coverage. Asymmetric and broad distributions of binding energies were found, with a maximum population peaking at low energy. We propose a model for the desorption kinetics that assumes a complete thermal equilibrium of the molecules with the ice film. The sample is characterized by a distribution of adsorption sites that are filled according to a Fermi-Dirac statistic law. The TPD curves can be simulated and fitted to provide the parameters describing the distribution of the molecules as a function of their binding energy. This approach contributes to a correct description of the interaction of molecular hydrogen with the surface of possibly porous grain mantles in the interstellar medium.

The Langmuir isotherm: a commonly applied but misleading approach for the analysis of protein adsorption behavior.

PubMed

Latour, Robert A

2015-03-01

The Langmuir adsorption isotherm provides one of the simplest and most direct methods to quantify an adsorption process. Because isotherm data from protein adsorption studies often appear to be fit well by the Langmuir isotherm model, estimates of protein binding affinity have often been made from its use despite that fact that none of the conditions required for a Langmuir adsorption process may be satisfied for this type of application. The physical events that cause protein adsorption isotherms to often provide a Langmuir-shaped isotherm can be explained as being due to changes in adsorption-induced spreading, reorientation, clustering, and aggregation of the protein on a surface as a function of solution concentration in contrast to being due to a dynamic equilibrium adsorption process, which is required for Langmuir adsorption. Unless the requirements of the Langmuir adsorption process can be confirmed, fitting of the Langmuir model to protein adsorption isotherm data to obtain thermodynamic properties, such as the equilibrium constant for adsorption and adsorption free energy, may provide erroneous values that have little to do with the actual protein adsorption process, and should be avoided. In this article, a detailed analysis of the Langmuir isotherm model is presented along with a quantitative analysis of the level of error that can arise in derived parameters when the Langmuir isotherm is inappropriately applied to characterize a protein adsorption process. © 2014 Wiley Periodicals, Inc.

A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures.

PubMed

Schellman, J A

1990-08-31

The properties of a simple model for solvation in mixed solvents are explored in this paper. The model is based on the supposition that solvent replacement is a simple one-for-one substitution reaction at macromolecular sites which are independent of one another. This leads to a new form for the binding polynomial in which all terms are associated with ligand interchange rather than ligand addition. The principal solvent acts as one of the ligands. Thermodynamic analysis then shows that thermodynamic binding (i.e., selective interaction) depends on the properties of K'-1, whereas stoichiometric binding (site occupation) depends on K'. K' is a 'practical' interchange equilibrium constant given by (f3/f1)K, where K is the true equilibrium constant for the interchange of components 3 and 1 on the site and f3 and f4 denote their respective activity coefficients on the mole fraction scale. Values of K' less than unity lead to negative selective interaction. It is selective interaction and not occupation number which determines the thermodynamic effects of solvation. When K' greater than 100 on the mole fraction scale or K' greater than 2 on the molality scale (in water), the differences between stoichiometric binding and selective interaction become less than 1%. The theory of this paper is therefore necessary only for very weak binding constants. When K'-1 is small, large concentrations of the added solvent component are required to produce a thermodynamic effect. Under these circumstances the isotherms for the selective interaction and for the excess (or transfer) free energy are strongly dependent on the behavior of the activity coefficients of both solvent components. Two classes of behavior are described depending on whether the components display positive or negative deviations from Raoult's law. Examples which are discussed are aqueous solutions of urea and guanidinium chloride for positive deviations and of sucrose and glucose for negative deviations. Examination of the few studies which have been reported in the literature shows that most of the qualitative features of the stabilization of proteins by sugars and their destabilization by urea and guanidinium chloride are faithfully represented with the model. This includes maxima in the free energy of stabilization and destabilization, decreased and zero selective interaction at high concentrations, etc. These phenomena had no prior explanation. Deficiencies in the model as a representation of solvation in aqueous solution are discussed in the appendix.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landefeld, T.D.; Byrne, M.D.; Campbell, K.L.

1981-12-01

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and themore » resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.« less

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

Spectro Analytical, Computational and In Vitro Biological Studies of Novel Substituted Quinolone Hydrazone and it's Metal Complexes.

PubMed

Nagula, Narsimha; Kunche, Sudeepa; Jaheer, Mohmed; Mudavath, Ravi; Sivan, Sreekanth; Ch, Sarala Devi

2018-01-01

Some novel transition metal [Cu (II), Ni (II) and Co (II)] complexes of nalidixic acid hydrazone have been prepared and characterized by employing spectro-analytical techniques viz: elemental analysis, 1 H-NMR, Mass, UV-Vis, IR, TGA-DTA, SEM-EDX, ESR and Spectrophotometry studies. The HyperChem 7.5 software was used for geometry optimization of title compound in its molecular and ionic forms. Quantum mechanical parameters, contour maps of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) and corresponding binding energy values were computed using semi empirical single point PM3 method. The stoichiometric equilibrium studies of metal complexes carried out spectrophotometrically using Job's continuous variation and mole ratio methods inferred formation of 1:2 (ML 2 ) metal complexes in respective systems. The title compound and its metal complexes screened for antibacterial and antifungal properties, exemplified improved activity in metal complexes. The studies of nuclease activity for the cleavage of CT- DNA and MTT assay for in vitro cytotoxic properties involving metal complexes exhibited high activity. In addition, the DNA binding properties of Cu (II), Ni (II) and Co (II) complexes investigated by electronic absorption and fluorescence measurements revealed their good binding ability and commended agreement of K b values obtained from both the techniques. Molecular docking studies were also performed to find the binding affinity of synthesized compounds with DNA (PDB ID: 1N37) and "Thymidine phosphorylase from E.coli" (PDB ID: 4EAF) protein targets.

Modulation of the platelet serotonin transporter by thermal balneotherapy: a study in healthy subjects.

PubMed

Baroni, S; Marazziti, D; Consoli, G; Picchetti, M; Catena-Dell'Osso, M; Galassi, A

2012-05-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 30 healthy volunteers before (t0) and 30 minutes after (t1) thermal balneotherapy with ozonized water, as compared with a similar group who underwent a bath in non-mineral water. MATERIALS AN METHODS: The SERT was evaluated by means of the specific binding of 3H-paroxetine (3H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of 3H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Thermal balneotherapy induces changes of the platelet serotonin transporter in healthy subjects.

PubMed

Marazziti, Donatella; Baroni, Stefano; Giannaccini, Gino; Catena Dell'Osso, Mario; Consoli, Giorgio; Picchetti, Michela; Carlini, Marina; Massimetti, Gabriele; Provenzano, Serafina; Galassi, Antonio

2007-10-01

Although the beneficial effects of balneotherapy have been recognized since a long time, a few information is available on the biological mechanisms underlying them and the subjective feelings of increased well-being and mood. The links between the serotonin (5-HT) system and mood prompted us to investigate the 5-HT platelet transporter (SERT), which is considered a reliable, peripheral marker of the same structure present in presynaptic neurons, in 20 healthy volunteers before (t0) and 30 min after (t1) thermal balneotherapy with ozonized water of Montecatini spa, as compared with a similar group who underwent a bath in non-mineral water. The SERT was evaluated by means of the specific binding of (3)H-paroxetine ((3)H-Par) to platelet membranes. Equilibrium-saturation binding data, the maximal binding capacity (Bmax) and the dissociation constant (Kd), were obtained by means of the Scatchard analysis. The results showed that, while Bmax values did not change in both groups, the Kd values decreased significantly at t1 only in those subjects who bathed in ozonized water. The results of this study, while showing a decrease of the dissociation constant (Kd) which is the inverse of affinity constant, of (3)H-Par binding to SERT in all subjects after balneotherapy and not in those bathing in normal water, suggest that SERT modifications may be related to a specific effect of ozonized water and, perhaps, also to the increased sense of well-being.

Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations

PubMed Central

Weinkam, Patrick; Sali, Andrej

2014-01-01

Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation. PMID:23957820

Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

PubMed

Wei, A P; Blumenthal, D K; Herron, J N

1994-05-01

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Understanding the Structural Ensembles of a Highly Extended Disordered Protein†

PubMed Central

Daughdrill, Gary W.; Kashtanov, Stepan; Stancik, Amber; Hill, Shannon E.; Helms, Gregory; Muschol, Martin

2013-01-01

Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble. PMID:21979461

ESI-MS measurements for the equilibrium constants of copper(II)-insulin complexes.

PubMed

Gülfen, Mustafa; Özdemir, Abdil; Lin, Jung-Lee; Chen, Chung-Hsuan

2018-06-01

Trace elements regulate many biological reactions in the body. Copper(II) is known as one of trace elements and capable of binding to proteins. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic β-cells. In this study, Cu(II)-insulin complexes were investigated by using ESI-MS method. Insulin molecule gives ESI-MS peaks at +4, +5, +6 and +7 charged states. Cu(II)-insulin complexes can be monitored and quantified on the ESI-MS spectra as the shifted peaks according to insulin peaks. The solutions of Cu(II)-insulin complexes at different pHs and mole ratios of Cu(II) ions to insulin molecule were measured on the ESI-MS. The highest complex formation ratio for Cu(II)-insulin were found at pH 7. The multiple bindings of Cu(II) ions to insulin molecule was observed. The formation equilibrium constants of Cu(II)-insulin complexes were calculated as Kf 1 : 3.34 × 10 4 , Kf 2 : 2.99 × 10 4 , Kf 3 : 7.00 × 10 3 and Kf 4 :2.86 × 10 3 . The specific binding property of Cu(II) ions was controlled by using different spray ion sources including electrospray and nano-electrospray. The binding property of Cu(II) also investigated by MS/MS fragmentation. It was concluded from the ESI-MS measurements that Cu(II) ion has a high affinity to insulin molecules to form stable complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Drug-DNA interactions at single molecule level: A view with optical tweezers

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan

Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by stretching the DNA molecule to a certain force and holding it at constant force while adding the drug and then while washing off the drug. Our finding resolves the long lasting controversy of ActD binding modes, clearly showing that both the dsDNA binding and ssDNA binding converge to the same single mode. The result supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by binding to transcription bubbles and causing cell death.

One Crystal, Two Temperatures: Cryocooling Penalties Alter Ligand Binding to Transient Protein Sites

DOE PAGES

Fischer, Marcus; Shoichet, Brian K.; Fraser, James S.

2015-05-28

Interrogating fragment libraries by X-ray crystallography is a powerful strategy for discovering allosteric ligands for protein targets. Cryocooling of crystals should theoretically increase the fraction of occupied binding sites and decrease radiation damage. However, it might also perturb protein conformations that can be accessed at room temperature. Using data from crystals measured consecutively at room temperature and at cryogenic temperature, we found that transient binding sites could be abolished at the cryogenic temperatures employed by standard approaches. Finally, changing the temperature at which the crystallographic data was collected could provide a deliberate perturbation to the equilibrium of protein conformations andmore » help to visualize hidden sites with great potential to allosterically modulate protein function.« less

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

PubMed

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

2009-12-18

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

Improved flow cytometer measurement of binding assays

DOEpatents

Saunders, G.C.

1984-05-30

The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

Dynamic Disorder in Quasi-Equilibrium Enzymatic Systems

PubMed Central

Chaudhury, Srabanti; Igoshin, Oleg A.

2010-01-01

Conformations and catalytic rates of enzymes fluctuate over a wide range of timescales. Despite these fluctuations, there exist some limiting cases in which the enzymatic catalytic rate follows the macroscopic rate equation such as the Michaelis-Menten law. In this paper we investigate the applicability of macroscopic rate laws for fluctuating enzyme systems in which catalytic transitions are slower than ligand binding-dissociation reactions. In this quasi-equilibrium limit, for an arbitrary reaction scheme we show that the catalytic rate has the same dependence on ligand concentrations as obtained from mass-action kinetics even in the presence of slow conformational fluctuations. These results indicate that the timescale of conformational dynamics – no matter how slow – will not affect the enzymatic rate in quasi-equilibrium limit. Our numerical results for two enzyme-catalyzed reaction schemes involving multiple substrates and inhibitors further support our general theory. PMID:20808776

Model-based analysis of coupled equilibrium-kinetic processes: indirect kinetic studies of thermodynamic parameters using the dynamic data.

PubMed

Emami, Fereshteh; Maeder, Marcel; Abdollahi, Hamid

2015-05-07

Thermodynamic studies of equilibrium chemical reactions linked with kinetic procedures are mostly impossible by traditional approaches. In this work, the new concept of generalized kinetic study of thermodynamic parameters is introduced for dynamic data. The examples of equilibria intertwined with kinetic chemical mechanisms include molecular charge transfer complex formation reactions, pH-dependent degradation of chemical compounds and tautomerization kinetics in micellar solutions. Model-based global analysis with the possibility of calculating and embedding the equilibrium and kinetic parameters into the fitting algorithm has allowed the complete analysis of the complex reaction mechanisms. After the fitting process, the optimal equilibrium and kinetic parameters together with an estimate of their standard deviations have been obtained. This work opens up a promising new avenue for obtaining equilibrium constants through the kinetic data analysis for the kinetic reactions that involve equilibrium processes.

Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E.

PubMed

Khan, Parvez; Prakash, Amresh; Haque, Md Anzarul; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

2016-10-01

Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F346) and CD signal at 222nm (θ222) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (Cm) values at 2.88M and 4.95M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔGD(0)) associated with the equilibrium of denaturation exist between native state↔denatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60ns was also performed. A clear correspondence was established between the in vitro and in silico studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Bench-Scale Testing and Process Performance Projections of CO2 Capture by CO2–Binding Organic Liquids (CO2BOLs) With and Without Polarity-Swing-Assisted Regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Feng; Heldebrant, David J.; Mathias, Paul M.

This manuscript provides a detailed analysis of a continuous flow, bench scale study of the CO2BOL solvent platform with and without its Polarity Swing Assisted Regeneration (PSAR). This study encompassed four months of continuous flow testing of a candidate CO2BOL with a thermal regeneration and PSAR regeneration using decane antisolvent. In both regeneration schemes, steady state capture of >90 %CO2 was achieved using simulated flue gas at acceptable L/G ratios. Aspen Plus™ modeling was performed to assess process performance compared to previous equilibrium performance projections. This paper also includes net power projections, and comparisons to DOE’s Case 10 amine baseline.

Combinatoric analysis of heterogeneous stochastic self-assembly.

PubMed

D'Orsogna, Maria R; Zhao, Bingyu; Berenji, Bijan; Chou, Tom

2013-09-28

We analyze a fully stochastic model of heterogeneous nucleation and self-assembly in a closed system with a fixed total particle number M, and a fixed number of seeds Ns. Each seed can bind a maximum of N particles. A discrete master equation for the probability distribution of the cluster sizes is derived and the corresponding cluster concentrations are found using kinetic Monte-Carlo simulations in terms of the density of seeds, the total mass, and the maximum cluster size. In the limit of slow detachment, we also find new analytic expressions and recursion relations for the cluster densities at intermediate times and at equilibrium. Our analytic and numerical findings are compared with those obtained from classical mass-action equations and the discrepancies between the two approaches analyzed.

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives.

PubMed

Pavlov, V; Kong Thoo Lin, P; Rodilla, V

2001-07-31

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC(50) values of 1.38, 2.91 and 8.45 microM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods (C(50) 0.08-0.12 microM) and their apparent equilibrium binding constants (K(app)) were calculated to be in the range of 10.5-18 x 10(7) M(-1). Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis (T(m)=93.3-97 degrees C compared with 75 degrees C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.

Kinetics of CO2 diffusion in human carbonic anhydrase: a study using molecular dynamics simulations and the Markov-state model.

PubMed

Chen, Gong; Kong, Xian; Lu, Diannan; Wu, Jianzhong; Liu, Zheng

2017-05-10

Molecular dynamics (MD) simulations, in combination with the Markov-state model (MSM), were applied to probe CO 2 diffusion from an aqueous solution into the active site of human carbonic anhydrase II (hCA-II), an enzyme useful for enhanced CO 2 capture and utilization. The diffusion process in the hydrophobic pocket of hCA-II was illustrated in terms of a two-dimensional free-energy landscape. We found that CO 2 diffusion in hCA-II is a rate-limiting step in the CO 2 diffusion-binding-reaction process. The equilibrium distribution of CO 2 shows its preferential accumulation within a hydrophobic domain in the protein core region. An analysis of the committors and reactive fluxes indicates that the main pathway for CO 2 diffusion into the active site of hCA-II is through a binding pocket where residue Gln 136 contributes to the maximal flux. The simulation results offer a new perspective on the CO 2 hydration kinetics and useful insights toward the development of novel biochemical processes for more efficient CO 2 sequestration and utilization.

Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant.

PubMed

Deng, Ge; Wu, Kristi; Cruce, Alex A; Bowman, Michael K; Vincent, John B

2015-02-01

Transferrin, the major iron transport protein in the blood, also transports trivalent chromium in vivo. Recent in vitro studies have, however, suggested that the binding of chromic ions to apotransferrin is too slow to be biologically relevant. Nevertheless, the in vitro studies have generally failed to adequately take physiological bicarbonate concentrations into account. In aqueous buffer (with ambient (bi)carbonate concentrations), the binding of chromium to transferrin is too slow to be physiologically relevant, taking days to reach equilibrium with the protein's associated conformational changes. However, in the presence of 25mM (bi)carbonate, the concentration in human blood, chromic ions bind rapidly and tightly to transferrin. Details of the kinetics of chromium binding to human serum transferrin and conalbumin (egg white transferrin) in the presence of bicarbonate and other major potential chromium ligands are described and are consistent with transferrin being the major chromic ion transporter from the blood to tissues. Copyright © 2014 Elsevier Inc. All rights reserved.

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination.

PubMed

Ahmed, M Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V; Gurevich, Eugenia V

2011-05-10

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions. PMID:23284658

Use of sunlight to partially detoxify groundnut (peanut) cake flour and casein contaminated with aflatoxin B1.

PubMed

Shantha, T; Murthy, V S

1981-03-01

Sunlight destroyed 83 and 50% of the toxin added to casein and groundnut cake flour, respectively. Equilibrium dialysis revealed that both casein and groundnut protein bind aflatoxin but the toxin bound to casein appeared more photo-labile than that bound to groundnut protein.

Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albumin.

PubMed

Sun, Shuangjiao; Long, Chanjuan; Tao, Chunyao; Meng, Sa; Deng, Biyang

2014-12-03

The paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL) for studying the interaction between human serum albumin (HSA) and trimetazidine dihydrochloride (TMZ). The time required for equilibrium by ultrasonic microdialysis was 45min, which was far less than that by traditional dialysis (240min). It took 80min to achieve the required combination equilibrium by normal incubation and only 20min by ultrasonic. Compared with traditional dialysis, the use of ultrasonic microdialysis simplified experimental procedures, shortened experimental time and saved consumption of sample. A simple, sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized. Under the optimized conditions, the linear range of TMZ was from 0.075 to 80μmol/L (r(2)=0.9974). The detection limit was 26nmol/L with RSD of 2.8%. The number of binding sites and binding constant were 1.54 and 15.17L/mol, respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

Fungal melanins and their interactions with metals.

PubMed

Fogarty, R V; Tobin, J M

1996-09-01

Fungal melanins are dark brown or black pigments located in cell walls. They also exist as extracellular polymers. Melanized fungi possess increased virulence and resistance to microbial attack as well as enhanced survival while under environmental stress. Melanins contain various functional groups which provide an array of multiple nonequivalent binding sites for metal ions. Pigmented Cladosporium cladosporoides was shown to biosorb 2.5- to four-fold more Ni, Cu, Zn, Cd, and Pb than albino Penicillium digitatum and at four- to six-fold higher rates. Metal desorption was significantly lower for extracellular melanin than from pigmented or albino biomass which indicated the strength of the melanin-metal bond. At equilibrium, tributyltin chloride (TBTC) concentrations of 2.5 mM, pigmented and albino Aureobasidium pullulans absorbed approximately 0.9 and 0.7 mumol TBTC mg -1 dry wt, respectively, whereas purified extracellular melanin exhibited uptake levels of approximately 22 mumol TBTC mg-1 dry wt at an equilibrium concentration of only 0.4 mM. Addition of melanin to the growth medium reduced the toxic effect of CuSO4 and TBTC due to melanin metal binding and sequestration.

Frequency Modulation of Transcriptional Bursting Enables Sensitive and Rapid Gene Regulation.

PubMed

Li, Congxin; Cesbron, François; Oehler, Michael; Brunner, Michael; Höfer, Thomas

2018-04-25

Gene regulation is a complex non-equilibrium process. Here, we show that quantitating the temporal regulation of key gene states (transcriptionally inactive, active, and refractory) provides a parsimonious framework for analyzing gene regulation. Our theory makes two non-intuitive predictions. First, for transcription factors (TFs) that regulate transcription burst frequency, as opposed to amplitude or duration, weak TF binding is sufficient to elicit strong transcriptional responses. Second, refractoriness of a gene after a transcription burst enables rapid responses to stimuli. We validate both predictions experimentally by exploiting the natural, optogenetic-like responsiveness of the Neurospora GATA-type TF White Collar Complex (WCC) to blue light. Further, we demonstrate that differential regulation of WCC target genes is caused by different gene activation rates, not different TF occupancy, and that these rates are tuned by both the core promoter and the distance between TF-binding site and core promoter. In total, our work demonstrates the relevance of a kinetic, non-equilibrium framework for understanding transcriptional regulation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Conformational and hydration properties modulate ice recognition by type I antifreeze protein and its mutants.

PubMed

Chakraborty, Sandipan; Jana, Biman

2017-05-10

The mechanism of ice recognition by antifreeze protein (AFP) is a topic of recent interest. Here, using equilibrium simulations and free energy calculations, we provide structural rationale to the observed experimental anomalies on type I AFP (wfAFP isoform HPLC6) and its mutants as well as probe the molecular origin of ice recognition by them. Our results clearly demonstrate that the interplay between the conformational and hydration properties dictates the ice binding ability of type I AFP and its mutants. We find that HPLC6 exists as a highly stable long helix which adsorbs on the ice surface through the ordered water cages around the CH 3 group of threonine (THR) residues, rather than directly binding to the ice surface via threonine (THR) through hydrogen bonding. Upon mutating THR with serine (SER), the straight helix conformation of HPLC6 disappears and the most stable conformation is a kinked helix devoid of ice binding ability. Free energy calculations reveal that there is a dynamic equilibrium between straight and bent helical conformations in the case of a valine (VAL) mutant. The straight long helical form of the VAL mutant also has the ability to form an ordered water cage structure around the CH 3 groups of the VAL residues and thereby efficiently adsorbs on an ice plane similar to the wild type AFP.

Antidiabetic Bis-Maltolato-OxoVanadium(IV): Conversion of inactive trans- to bioactive cis-BMOV for possible binding to target PTP-1B

PubMed Central

Scior, Thomas; Mack, Hans-Georg; García, José Antonio Guevara; Koch, Wolfhard

2008-01-01

The postulated transition of Bis-Maltolato-OxoVanadium(IV) (BMOV) from its inactive trans- into its cis-aquo-BMOV isomeric form in solution was simulated by means of computational molecular modeling. The rotational barrier was calculated with DFT – B3LYP under a stepwise optimization protocol with STO-3G, 3-21G, 3-21G*, and 6-31G ab initio basis sets. Our computed results are consistent with reports on the putative molecular mechanism of BMOV triggering the insulin-like cellular response (insulin mimetic) as a potent inhibitor of the protein tyrosine phosphatase-1B (PTP-1B). Initially, trans-BMOV is present in its solid dosage form but in aqueous solution, and during oral administration, it is readily converted into a mixture of “open-type” and “closed-type” complexes of cis-aquo-BMOV under equilibrium conditions. However, in the same measure as the “closed-type” complex binds to the cytosolic PTP-1B, it disappears from solution, and the equilibrium shifts towards the “closed-type” species. In full accordance, the computed binding mode of cis-BMOV is energetically favored over sterically hindered trans-BMOV. In view of our earlier report on prodrug hypothesis of vanadium organic compounds the present results suggest that cis-BMOV is the bioactive species. PMID:19920909

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fei, Yiyan; Landry, James P.; Zhu, X. D., E-mail: xdzhu@physics.ucdavis.edu

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400–10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven,more » entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.« less

Photocaged Competitor Guests: A General Approach Toward Light-Activated Cargo Release From Cucurbiturils.

PubMed

Romero, Miguel A; Basílio, Nuno; Moro, Artur J; Domingues, Mara; González-Delgado, José A; Arteaga, Jesús F; Pischel, Uwe

2017-09-21

A general approach toward the light-induced guest release from cucurbit[7]uril by means of a photoactivatable competitor was devised. An o-nitrobenzyl-caged competitor is photolyzed to generate a competitive guest that can displace cargo from the host macrocycle solely based on considerations of chemical equilibrium. With this method the release of terpene guests from inclusion complexes with cucurbit[7]uril was demonstrated. The binding of the herein investigated terpenes, all being lead fragrant components in essential oils, has been characterized for the first time. They feature binding constants of up to 10 8  L mol -1 and a high differential binding selectivity (spanning four orders of magnitude for the binding constants for the particular set of terpenes). By fine-tuning the photoactivatable competitor guest, selective and also sequential release of the terpenes was achieved. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Self-Organization of Metal Nanoparticles in Light: Electrodynamics-Molecular Dynamics Simulations and Optical Binding Experiments.

PubMed

McCormack, Patrick; Han, Fei; Yan, Zijie

2018-02-01

Light-driven self-organization of metal nanoparticles (NPs) can lead to unique optical matter systems, yet simulation of such self-organization (i.e., optical binding) is a complex computational problem that increases nonlinearly with system size. Here we show that a combined electrodynamics-molecular dynamics simulation technique can simulate the trajectories and predict stable configurations of silver NPs in optical fields. The simulated dynamic equilibrium of a two-NP system matches the probability density of oscillations for two optically bound NPs obtained experimentally. The predicted stable configurations for up to eight NPs are further compared to experimental observations of silver NP clusters formed by optical binding in a Bessel beam. All configurations are confirmed to form in real systems, including pentagonal clusters with five-fold symmetry. Our combined simulations and experiments have revealed a diverse optical matter system formed by anisotropic optical binding interactions, providing a new strategy to discover artificial materials.

Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

PubMed

Sultana, Azmiri; Lee, Jeffrey E

2015-02-02

Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. Copyright © 2015 John Wiley & Sons, Inc.

Quantitation of Membrane-Ligand Interactions Using Backscattering Interferometry

PubMed Central

Baksh, Michael M.; Kussrow, Amanda K.; Mileni, Mauro; Finn, M.G.; Bornhop, Darryl J.

2011-01-01

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest. PMID:21399645

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

NASA Astrophysics Data System (ADS)

Fei, Yiyan; Landry, James P.; Li, Yanhong; Yu, Hai; Lau, Kam; Huang, Shengshu; Chokhawala, Harshal A.; Chen, Xi; Zhu, X. D.

2013-11-01

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400-10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven, entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells*

PubMed Central

Okamura, Tatsunori; Antoun, Gamil; Keir, Stephen T.; Friedman, Henry; Bigner, Darell D.; Ali-Osman, Francis

2015-01-01

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs. PMID:26429914

Cytosolic proteins can exploit membrane localization to trigger functional assembly

PubMed Central

2018-01-01

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10–1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding. PMID:29505559

Application of the Nernst-Planck approach to lead ion exchange in Ca-loaded Pelvetia canaliculata.

PubMed

Costa, Joana F de Sá S; Vilar, Vítor J P; Botelho, Cidália M S; da Silva, Eduardo A B; Boaventura, Rui A R

2010-07-01

Ca-loaded Pelvetia canaliculata biomass was used to remove Pb(2+) in aqueous solution from batch and continuous systems. The physicochemical characterization of algae Pelvetia particles by potentiometric titration and FTIR analysis has shown a gel structure with two major binding groups - carboxylic (2.8 mmol g(-1)) and hydroxyl (0.8 mmol g(-1)), with an affinity constant distribution for hydrogen ions well described by a Quasi-Gaussian distribution. Equilibrium adsorption (pH 3 and 5) and desorption (eluents: HNO(3) and CaCl(2)) experiments were performed, showing that the biosorption mechanism was attributed to ion exchange among calcium, lead and hydrogen ions with stoichiometry 1:1 (Ca:Pb) and 1:2 (Ca:H and Pb:H). The uptake capacity of lead ions decreased with pH, suggesting that there is a competition between H(+) and Pb(2+) for the same binding sites. A mass action law for the ternary mixture was able to predict the equilibrium data, with the selectivity constants alpha(Ca)(H)=9+/-1 and alpha(Ca)(Pb)=44+/-5, revealing a higher affinity of the biomass towards lead ions. Adsorption (initial solution pH 4.5 and 2.5) and desorption (0.3M HNO(3)) kinetics were performed in batch and continuous systems. A mass transfer model using the Nernst-Planck approximation for the ionic flux of each counter-ion was used for the prediction of the ions profiles in batch systems and packed bed columns. The intraparticle effective diffusion constants were determined as 3.73x10(-7)cm(2)s(-1) for H(+), 7.56x10(-8)cm(2)s(-1) for Pb(2+) and 6.37x10(-8)cm(2)s(-1) for Ca(2+). Copyright 2010 Elsevier Ltd. All rights reserved.

Thermodynamic analysis of the disorder-to-α-helical transition of 18.5-kDa myelin basic protein reveals an equilibrium intermediate representing the most compact conformation.

PubMed

Vassall, Kenrick A; Jenkins, Andrew D; Bamm, Vladimir V; Harauz, George

2015-05-22

The intrinsically disordered, 18.5-kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that is essential to proper myelin formation in the central nervous system. MBP acts in oligodendrocytes both to adjoin membrane leaflets to each other in forming myelin and as a hub in numerous protein-protein and protein-membrane interaction networks. Like many intrinsically disordered proteins (IDPs), MBP multifunctionality arises from its high conformational plasticity and its ability to undergo reversible disorder-to-order transitions. One such transition is the disorder-to-α-helical conformational change that is induced upon MBP-membrane binding. Here, we have investigated the disorder-to-α-helical transition of MBP-derived α-peptides and the full-length 18.5-kDa protein. This transition was induced through titration of the membrane-mimetic solvent trifluoroethanol into both protein and peptide solutions, and conformational change was monitored using circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid binding, tryptophan fluorescence quenching, and Förster (fluorescence) resonance energy transfer measurements. The data suggest that the disorder-to-α-helical transition of MBP follows a 3-state model: disordered↔intermediate↔α-helical, with each of the identified equilibrium states likely representing a conformational ensemble. The disordered state is characterized by slight compaction with little regular secondary structure, whereas the intermediate is also disordered but globally more compact. Surprisingly, the α-helical conformation is less compact than the intermediate. This study suggests that multifunctionality in MBP could arise from differences in the population of energetically distinct ensembles under different conditions and also provides an example of an IDP that undergoes cooperative global conformation change. Copyright © 2015 Elsevier Ltd. All rights reserved.

Thermodynamic and kinetic analyses of curcumin and bovine serum albumin binding.

PubMed

Hudson, Eliara Acipreste; de Paula, Hauster Maximiler Campos; Ferreira, Guilherme Max Dias; Ferreira, Gabriel Max Dias; Hespanhol, Maria do Carmo; da Silva, Luis Henrique Mendes; Pires, Ana Clarissa Dos S

2018-03-01

Bovine serum albumin (BSA)/curcumin binding and dye photodegradation stability were evaluated. BSA/curcumin complex showed 1:1 stoichiometry, but the thermodynamic binding parameters depended on the technique used and BSA conformation. The binding constant was of the order of 10 5 L·mol -1 by fluorescence and microcalorimetric, and 10 3 and 10 4 L·mol -1 by surface plasmon resonance (steady-state equilibrium and kinetic experiments, respectively). For native BSA/curcumin, fluorescence indicated an enthalpic and entropic driven process based on the standard enthalpy change (ΔH ○ F =-8.67kJ·mol -1 ), while microcalorimetry showed an entropic driven binding process (ΔH ○ cal =29.11kJ·mol -1 ). For the unfolded BSA/curcumin complex, it was found thatp ΔH ○ F =-16.12kJ·mol -1 and ΔH ○ cal =-42.63kJ·mol -1 . BSA (mainly native) increased the curcumin photodegradation stability. This work proved the importance of using different techniques to characterize the protein-ligand binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Binding of Dissolved Strontium by Micrococcus luteus

PubMed Central

Faison, Brendlyn D.; Cancel, Carmen A.; Lewis, Susan N.; Adler, Howard I.

1990-01-01

Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl2 at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H+. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity. PMID:16348370

Improved flow cytometer measurement of binding assays

NASA Astrophysics Data System (ADS)

Saunders, G. C.

1984-05-01

A method of measuring binding assays is carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also known quantity of smaller particles with a coating of binder reactant. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating.

Calorimetric and spectroscopic investigation of the interaction between the C-terminal domain of Enzyme I and its ligands

PubMed Central

Yun, Young-Joo; Suh, Jeong-Yong

2012-01-01

Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (KD) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with KD of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain–domain motion in EI can contribute as large as ∼−3.2 kcal/mol toward PEP binding. PMID:22936614

Non-equilibrium STLS approach to transport properties of single impurity Anderson model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rezai, Raheleh, E-mail: R_Rezai@sbu.ac.ir; Ebrahimi, Farshad, E-mail: Ebrahimi@sbu.ac.ir

In this work, using the non-equilibrium Keldysh formalism, we study the effects of the electron–electron interaction and the electron-spin correlation on the non-equilibrium Kondo effect and the transport properties of the symmetric single impurity Anderson model (SIAM) at zero temperature by generalizing the self-consistent method of Singwi, Tosi, Land, and Sjolander (STLS) for a single-band tight-binding model with Hubbard type interaction to out of equilibrium steady-states. We at first determine in a self-consistent manner the non-equilibrium spin correlation function, the effective Hubbard interaction, and the double-occupancy at the impurity site. Then, using the non-equilibrium STLS spin polarization function in themore » non-equilibrium formalism of the iterative perturbation theory (IPT) of Yosida and Yamada, and Horvatic and Zlatic, we compute the spectral density, the current–voltage characteristics and the differential conductance as functions of the applied bias and the strength of on-site Hubbard interaction. We compare our spectral densities at zero bias with the results of numerical renormalization group (NRG) and depict the effects of the electron–electron interaction and electron-spin correlation at the impurity site on the aforementioned properties by comparing our numerical result with the order U{sup 2} IPT. Finally, we show that the obtained numerical results on the differential conductance have a quadratic universal scaling behavior and the resulting Kondo temperature shows an exponential behavior. -- Highlights: •We introduce for the first time the non-equilibrium method of STLS for Hubbard type models. •We determine the transport properties of SIAM using the non-equilibrium STLS method. •We compare our results with order-U2 IPT and NRG. •We show that non-equilibrium STLS, contrary to the GW and self-consistent RPA, produces the two Hubbard peaks in DOS. •We show that the method keeps the universal scaling behavior and correct exponential behavior of Kondo temperature.« less

Free energy profiles of cocaine esterase-cocaine binding process by molecular dynamics and potential of mean force simulations.

PubMed

Zhang, Yuxin; Huang, Xiaoqin; Han, Keli; Zheng, Fang; Zhan, Chang-Guo

2016-11-25

The combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profile of the CocE)-(+)-cocaine binding process in comparison with that of the corresponding CocE-(-)-cocaine binding process. According to the MD simulations, the equilibrium CocE-(+)-cocaine binding mode is similar to the CocE-(-)-cocaine binding mode. However, based on the simulated free energy profiles, a significant free energy barrier (∼5 kcal/mol) exists in the CocE-(+)-cocaine binding process whereas no obvious free energy barrier exists in the CocE-(-)-cocaine binding process, although the free energy barrier of ∼5 kcal/mol is not high enough to really slow down the CocE-(+)-cocaine binding process. In addition, the obtained free energy profiles also demonstrate that (+)-cocaine and (-)-cocaine have very close binding free energies with CocE, with a negligible difference (∼0.2 kcal/mol), which is qualitatively consistent with the nearly same experimental K M values of the CocE enzyme for (+)-cocaine and (-)-cocaine. The consistency between the computational results and available experimental data suggests that the mechanistic insights obtained from this study are reasonable. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

PubMed Central

2015-01-01

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs. PMID:25226846

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Selective binding of pyrene in subdomain IB of human serum albumin: Combining energy transfer spectroscopy and molecular modelling to understand protein binding flexibility

NASA Astrophysics Data System (ADS)

Ling, Irene; Taha, Mohamed; Al-Sharji, Nada A.; Abou-Zied, Osama K.

2018-04-01

The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27 Å, which was closely reproduced by the docking analysis (29 Å) and MD simulation (32 Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I1/I3 of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5 nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding sites of the protein that can easily adapt their shape to accommodate a variety of molecular structures.

A novel substance P binding site in bovine adrenal medulla.

PubMed

Geraghty, D P; Livett, B G; Rogerson, F M; Burcher, E

1990-05-04

Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant (Kd) 1.46 +/- 0.73 nM, Bmax 0.73 +/- 0.06 pmol/g wet weight and Hill coefficient 0.98 +/- 0.01. Specific binding of [3H]SP was displaced by SP greater than neurokinin A (NKA) greater than SP(3-11) approximately SP(1-9) greater than SP(1-7) approximately SP(1-4) approximately SP(1-6), with neurokinin B (NKB) and SP(1-3) very weak competitors and SP(5-11), SP(7-11) and SP(9-11) causing negligible inhibition (up to 10 microM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys3 and/or Pro4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.

Anomalous versus slowed-down Brownian diffusion in the ligand-binding equilibrium.

PubMed

Soula, Hédi; Caré, Bertrand; Beslon, Guillaume; Berry, Hugues

2013-11-05

Measurements of protein motion in living cells and membranes consistently report transient anomalous diffusion (subdiffusion) that converges back to a Brownian motion with reduced diffusion coefficient at long times after the anomalous diffusion regime. Therefore, slowed-down Brownian motion could be considered the macroscopic limit of transient anomalous diffusion. On the other hand, membranes are also heterogeneous media in which Brownian motion may be locally slowed down due to variations in lipid composition. Here, we investigate whether both situations lead to a similar behavior for the reversible ligand-binding reaction in two dimensions. We compare the (long-time) equilibrium properties obtained with transient anomalous diffusion due to obstacle hindrance or power-law-distributed residence times (continuous-time random walks) to those obtained with space-dependent slowed-down Brownian motion. Using theoretical arguments and Monte Carlo simulations, we show that these three scenarios have distinctive effects on the apparent affinity of the reaction. Whereas continuous-time random walks decrease the apparent affinity of the reaction, locally slowed-down Brownian motion and local hindrance by obstacles both improve it. However, only in the case of slowed-down Brownian motion is the affinity maximal when the slowdown is restricted to a subregion of the available space. Hence, even at long times (equilibrium), these processes are different and exhibit irreconcilable behaviors when the area fraction of reduced mobility changes. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Strongly Interacting Fermi Gases: Non-Equilibrium Dynamics and Dimensional Crossover

NASA Astrophysics Data System (ADS)

Sommer, Ariel

2015-05-01

Strongly interacting atomic Fermi gases near Feshbach resonances give access to a rich variety of phenomena in many-fermion physics and superfluidity. This flexible and microscopically well-characterized system provides a pristine platform in which to benchmark many-body theories. I will describe three experiments on gases of fermionic 6Li atoms. In the first experiment, we study spin transport in the return to equilibrium after a spin excitation. From the dynamics near equilibrium, we obtain spin transport coefficients over a range of temperatures and interaction strengths, and observe quantum-limited spin diffusion at unitarity. In separate experiments, we study the effect of dimensionality on the binding of pairs of fermions. We tune the system from three to two dimensions by adjusting the strength of a one-dimensional optical lattice, and measure the binding energy of fermion pairs using radio-frequency spectroscopy. In a third set of experiments, we study nonlinear excitations of a fermionic superfluid. Imprinting a phase jump on the superfluid order parameter causes a long-lived, localized density depletion that oscillates through the cloud. We measure the oscillation period and find that it corresponds to an emergent particle with an effective mass of up to several hundred times the bare mass. This excitation has been identified as a solitonic vortex that results from the decay of a planar soliton. This work was performed at the Massachusetts Institute of Technology under the supervision of Prof. Martin Zwierlein.

Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

NASA Astrophysics Data System (ADS)

Marshall Mccall, Patrick

Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non-equilibrium turnover on the steady-state properties of collections of filaments remained unclear. Here, I reconstituted tunable, non-equilibrium actin turnover dynamics in entangled solutions of actin filaments as a model of the actin cortex of living cells. We found that this non-equilibrium turnover decouples solution mechanics from microstructure, enabling structurally indistinguishable materials to behave effectively as either viscous fluids or elastic gels. Additionally, we employed computer simulations to identify the dynamical regime in which actin turnover controls the effective viscosity of 2D cross-linked actin networks in the presence of motors. Additionally, I examine in this thesis the localization and self-assembly of actin filaments in condensed liquid phases called polyelectrolyte coacervates as a model membrane-less organelle. We find that concentration of actin through spontaneous partitioning preferentially to the coacervate phase accelerates the assembly of filaments. These filaments then localize to the coacervate-bulk interface, generating particles with visco-elastic shells surrounding liquid cores. In this case, the properties of the condensed phase enable regulation of actin assembly dynamics.

Basis for ligand discrimination between ON and OFF state riboswitch conformations: The case of the SAM-I riboswitch

PubMed Central

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-ela, Fareed

2012-01-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming “hybrid” structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding. PMID:22543867

Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.

PubMed

Ito, Takashi; Takahashi, Masayuki; Okazaki, Osamu; Sugiyama, Yuichi

2010-08-02

The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.

Basis for ligand discrimination between ON and OFF state riboswitch conformations: the case of the SAM-I riboswitch.

PubMed

Boyapati, Vamsi Krishna; Huang, Wei; Spedale, Jessica; Aboul-Ela, Fareed

2012-06-01

Riboswitches are RNA elements that bind to effector ligands and control gene expression. Most consist of two domains. S-Adenosyl Methionine (SAM) binds the aptamer domain of the SAM-I riboswitch and induces conformational changes in the expression domain to form an intrinsic terminator (transcription OFF state). Without SAM the riboswitch forms the transcription ON state, allowing read-through transcription. The mechanistic link between the SAM/aptamer recognition event and subsequent secondary structure rearrangement by the riboswitch is unclear. We probed for those structural features of the Bacillus subtilis yitJ SAM-I riboswitch responsible for discrimination between the ON and OFF states by SAM. We designed SAM-I riboswitch RNA segments forming "hybrid" structures of the ON and OFF states. The choice of segment constrains the formation of a partial P1 helix, characteristic of the OFF state, together with a partial antiterminator (AT) helix, characteristic of the ON state. For most choices of P1 vs. AT helix lengths, SAM binds with micromolar affinity according to equilibrium dialysis. Mutational analysis and in-line probing confirm that the mode of SAM binding by hybrid structures is similar to that of the aptamer. Altogether, binding measurements and in-line probing are consistent with the hypothesis that when SAM is present, stacking interactions with the AT helix stabilize a partially formed P1 helix in the hybrids. Molecular modeling indicates that continuous stacking between the P1 and the AT helices is plausible with SAM bound. Our findings raise the possibility that conformational intermediates may play a role in ligand-induced aptamer folding.

Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase.

PubMed

Hiller, R; Carmeli, C

1990-07-03

The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase

PubMed Central

Borders, Charles L.; MacGregor, Katherine M.; Edmiston, Paul L.; Gbeddy, Elikem R.K.; Thomenius, Michael J.; Mulligan, Guy B.; Snider, Mark J.

2003-01-01

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in kcat in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the KM values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in kcat. Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases. PMID:12592023

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase.

PubMed

Borders, Charles L; MacGregor, Katherine M; Edmiston, Paul L; Gbeddy, Elikem R K; Thomenius, Michael J; Mulligan, Guy B; Snider, Mark J

2003-03-01

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.

Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.

PubMed

Rangachari, Vijayaraghavan; Marin, Vedrana; Bienkiewicz, Ewa A; Semavina, Maria; Guerrero, Luis; Love, John F; Murphy, John R; Logan, Timothy M

2005-04-19

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Stability and electronic properties of oxygen-doped ZnS polytypes: DFTB study

NASA Astrophysics Data System (ADS)

Popov, Ilya S.; Vorokh, Andrey S.; Enyashin, Andrey N.

2018-06-01

Synthesis from aqueous solutions is an affordable method for fabrication of II-VI semiconductors. However, application of this method often imposes a disorder of crystal lattice, manifesting as a rich variety of polytypes arising from wurtzite and zinc blende phases. The origin of this disordering still remains debatable. Here, the influence of the most likely impurity at water environment - substitutional oxygen - on the polytypic equilibrium of zinc sulphide is studied by means of density-functional tight-binding method. According to calculations, the inclusion of such oxygen does not affect the polytypic equilibrium. Apart of thermodynamic stability, the electronic and elastic properties of ZnS polytypes are studied as the function of oxygen distribution.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knapp, R.J.; Sharma, S.D.; Toth, G.

(D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sitesmore » in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.« less

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. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A mass balance approach for calculation of recovery and binding enables the use of ultrafiltration as a rapid method for measurement of plasma protein binding for even highly lipophilic compounds.

PubMed

Wang, Changguang; Williams, Noelle S

2013-03-05

The aim of this study is to further validate the use of ultrafiltration (UF) as a method for determining plasma protein binding (PPB) by demonstrating that non-specific binding (NSB) is not a limitation, even for highly lipophilic compounds, because NSB sites on the apparatus are passivated in the presence of plasma. Mass balance theory was used to calculate recovery of 20 commercial and seven investigational compounds during ultrafiltration in the presence and absence of plasma. PPB was also measured using this mass balance approach for comparison to PPB determined by rapid equilibrium dialysis (RED) and as found in the literature. Compound recovery during UF was dramatically different in the presence and absence of plasma for compounds with high NSB in PBS only. A comparison of PPB calculated by ultrafiltration with literature values or calculated by RED gave concordant results. Discrepancies could be explained by changes in pH, insufficient time to equilibrium, or compound instability during RED, problems which were circumvented by ultrafiltration. Therefore, NSB, as measured by the traditional incubation of compound in PBS, need not be an issue when choosing UF as a PPB assay method. It is more appropriate to calculate compound recovery from the device in plasma as measured by mass balance to determine the suitability of the method for an individual compound. The speed with which UF can be conducted additionally avoids changes in pH or compound loss that can occur with other methods. The mass balance approach to UF is thus a preferred method for rapid determination of PPB. Copyright © 2012 Elsevier B.V. All rights reserved.

Allosteric monofunctional aspartate kinases from Arabidopsis.

PubMed

Curien, Gilles; Laurencin, Mathieu; Robert-Genthon, Mylène; Dumas, Renaud

2007-01-01

Plant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form.

Kinetic and equilibrium properties of regulatory Ca(2+)-binding domains in sodium-calcium exchangers 2 and 3.

PubMed

Tal, Inbal; Kozlovsky, Tom; Brisker, Dafna; Giladi, Moshe; Khananshvili, Daniel

2016-04-01

In mammals, three sodium-calcium exchanger (NCX) protein isoforms (NCX1, NCX2, and NCX3) mediate Ca(2+) fluxes across the membrane to maintain cellular Ca(2+) homeostasis. NCX isoforms and their splice variants are expressed in a tissue-specific manner to meet physiological demands. NCX1 is ubiquitously expressed, NCX2 is expressed in the brain and spinal cord, and NCX3 is expressed in the brain and skeletal muscle. Eukaryotic NCXs contain two cytosolic regulatory Ca(2+)-binding domains, CBD1 and CBD2, which form a two-domain tandem (CBD12) through a short linker. Ca(2+) binding to the CBDs underlies allosteric regulation of NCX. Previous structural and functional studies in NCX1 have shown that the CBDs synergistically interact, where their interactions are modulated in a splice variant-specific manner by splicing segment at CBD2. Here, we analyze the equilibrium and kinetic properties of Ca(2+) binding to purified preparations of CBD1, CBD2, and CBD12 from NCX2 and from NCX3 splice variants. We show that CBD1 interacts with CBD2 in the context of the CBD12 tandem in all NCX isoforms, where these interactions specifically modulate Ca(2+) sensing at the primary sensor of CBD1 to meet the physiological requirements. For example, the rate-limiting slow dissociation of "occluded" Ca(2+) from the primary allosteric sensor of variants expressed in skeletal muscle is ∼10-fold slower than that of variants expressed in the brain. Notably, these kinetic differences between NCX variants occur while maintaining a similar Ca(2+) affinity of the primary sensor, since the resting [Ca(2+)]i levels are similar among different cell types. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.

PubMed

Liao, Shu Y; Lee, Myungwoon; Hong, Mei

2018-03-01

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31 P NMR spectra and 1 H- 31 P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist. Copyright © 2018 Elsevier Inc. All rights reserved.

Bifurcation and Stability Analysis of the Equilibrium States in Thermodynamic Systems in a Small Vicinity of the Equilibrium Values of Parameters

NASA Astrophysics Data System (ADS)

Barsuk, Alexandr A.; Paladi, Florentin

2018-04-01

The dynamic behavior of thermodynamic system, described by one order parameter and one control parameter, in a small neighborhood of ordinary and bifurcation equilibrium values of the system parameters is studied. Using the general methods of investigating the branching (bifurcations) of solutions for nonlinear equations, we performed an exhaustive analysis of the order parameter dependences on the control parameter in a small vicinity of the equilibrium values of parameters, including the stability analysis of the equilibrium states, and the asymptotic behavior of the order parameter dependences on the control parameter (bifurcation diagrams). The peculiarities of the transition to an unstable state of the system are discussed, and the estimates of the transition time to the unstable state in the neighborhood of ordinary and bifurcation equilibrium values of parameters are given. The influence of an external field on the dynamic behavior of thermodynamic system is analyzed, and the peculiarities of the system dynamic behavior are discussed near the ordinary and bifurcation equilibrium values of parameters in the presence of external field. The dynamic process of magnetization of a ferromagnet is discussed by using the general methods of bifurcation and stability analysis presented in the paper.

Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

PubMed Central

Kim, J. Dongun; Senn, Stefan; Harel, Arye; Jelen, Benjamin I.; Falkowski, Paul G.

2013-01-01

Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth's history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet's surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this ‘composome’ analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity. PMID:23754810

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2.

PubMed

Decher, Niels; Gonzalez, Teresa; Streit, Anne Kathrin; Sachse, Frank B; Renigunta, Vijay; Soom, Malle; Heinemann, Stefan H; Daut, Jürgen; Sanguinetti, Michael C

2008-12-03

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.

An investigation of molecular dynamics simulation and molecular docking: interaction of citrus flavonoids and bovine β-lactoglobulin in focus.

PubMed

Sahihi, M; Ghayeb, Y

2014-08-01

Citrus flavonoids are natural compounds with important health benefits. The study of their interaction with a transport protein, such as bovine β-lactoglobulin (BLG), at the atomic level could be a valuable factor to control their transport to biological sites. In the present study, molecular docking and molecular dynamics simulation methods were used to investigate the interaction of hesperetin, naringenin, nobiletin and tangeretin as citrus flavonoids and BLG as transport protein. The molecular docking results revealed that these flavonoids bind in the internal cavity of BLG and the BLG affinity for binding the flavonoids follows naringenin>hesperetin>tangeretin>nobiletin. The docking results also indicated that the BLG-flavonoid complexes are stabilized through hydrophobic interactions, hydrogen bond interactions and π-π stacking interactions. The analysis of molecular dynamics (MD) simulation trajectories showed that the root mean square deviation (RMSD) of various systems reaches equilibrium and fluctuates around the mean value at various times. Time evolution of the radius of gyration, total solvent accessible surface of the protein and the second structure of protein showed as well that BLG and BLG-flavonoid complexes were stable around 2500ps, and there was not any conformational change as for BLG-flavonoid complexes. Further, the profiles of atomic fluctuations indicated the rigidity of the ligand binding site during the simulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water

PubMed Central

Chitpong, Nithinart; Husson, Scott M.

2016-01-01

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (Rh) measurements for PAA and PIA obtained from dynamic light scattering, which show that Rh values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration. PMID:27999394

Nanofiber Ion-Exchange Membranes for the Rapid Uptake and Recovery of Heavy Metals from Water.

PubMed

Chitpong, Nithinart; Husson, Scott M

2016-12-20

An evaluation of the performance of polyelectrolyte-modified nanofiber membranes was undertaken to determine their efficacy in the rapid uptake and recovery of heavy metals from impaired waters. The membranes were prepared by grafting poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) to cellulose nanofiber mats. Performance measurements quantified the dynamic ion-exchange capacity for cadmium (Cd), productivity, and recovery of Cd(II) from the membranes by regeneration. The dynamic binding capacities of Cd(II) on both types of nanofiber membrane were independent of the linear flow velocity, with a residence time of as low as 2 s. Analysis of breakthrough curves indicated that the mass flow rate increased rapidly at constant applied pressure after membranes approached equilibrium load capacity for Cd(II), apparently due to a collapse of the polymer chains on the membrane surface, leading to an increased porosity. This mechanism is supported by hydrodynamic radius (R h ) measurements for PAA and PIA obtained from dynamic light scattering, which show that R h values decrease upon Cd(II) binding. Volumetric productivity was high for the nanofiber membranes, and reached 0.55 mg Cd/g/min. The use of ethylenediaminetetraacetic acid as regeneration reagent was effective in fully recovering Cd(II) from the membranes. Ion-exchange capacities were constant over five cycles of binding-regeneration.

Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

PubMed

Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

2015-02-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

PubMed Central

Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

2014-01-01

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

PubMed

Bolla, Michela; Zenoni, Sara; Scheurer, Stephan; Vieths, Stefan; San Miguel Moncin, Maria Del Mar; Olivieri, Mario; Antico, Andrea; Ferrer, Marta; Berroa, Felicia; Enrique, Ernesto; Avesani, Linda; Marsano, Francesco; Zoccatelli, Gianni

2014-01-01

Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs. © 2014 S. Karger AG, Basel.

Curcuminoid Binding to Embryonal Carcinoma Cells: Reductive Metabolism, Induction of Apoptosis, Senescence, and Inhibition of Cell Proliferation

PubMed Central

Quitschke, Wolfgang W.

2012-01-01

Curcumin preparations typically contain a mixture of polyphenols, collectively referred to as curcuminoids. In addition to the primary component curcumin, they also contain smaller amounts of the co-extracted derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids can be differentially solubilized in serum, which allows for the systematic analysis of concentration-dependent cellular binding, biological effects, and metabolism. Technical grade curcumin was solubilized in fetal calf serum by two alternative methods yielding saturated preparations containing either predominantly curcumin (60%) or bisdemethoxycurcumin (55%). Continual exposure of NT2/D1 cells for 4–6 days to either preparation in cell culture media reduced cell division (1–5 µM), induced senescence (6–7 µM) or comprehensive cell death (8–10 µM) in a concentration-dependent manner. Some of these effects could also be elicited in cells transiently exposed to higher concentrations of curcuminoids (47 µM) for 0.5–4 h. Curcuminoids induced apoptosis by generalized activation of caspases but without nucleosomal fragmentation. The equilibrium binding of serum-solubilized curcuminoids to NT2/D1 cells incubated with increasing amounts of curcuminoid-saturated serum occurred with apparent overall dissociation constants in the 6–10 µM range. However, the presence of excess free serum decreased cellular binding in a hyperbolic manner. Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway. Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines. These results suggest that curcuminoids interact with cellular binding sites, thereby activating signal transduction pathways that initiate a variety of biological responses. The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites. Defined serum-solubilized curcuminoids used in cell culture media are thus suitable for further investigating the differential activation of signal transduction pathways. PMID:22768090

Spreading out of perturbations in reversible reaction networks

NASA Astrophysics Data System (ADS)

Maslov, Sergei; Sneppen, Kim; Ispolatov, I.

2007-08-01

Using an example of physical interactions between proteins, we study how a perturbation propagates in the equilibrium of a network of reversible reactions governed by the law of mass action. We introduce a matrix formalism to describe the linear response of all equilibrium concentrations to shifts in total abundances of individual reactants, and reveal its heuristic analogy to the flow of electric current in a network of resistors. Our main conclusion is that, on average, the induced changes in equilibrium concentrations decay exponentially as a function of network distance from the source of perturbation. We analyze how this decay is influenced by such factors as the topology of a network, binding strength, and correlations between concentrations of neighboring nodes. We find that the minimal branching of the network, small values of dissociation constants, and low equilibrium free (unbound) concentrations of reacting substances all decrease the decay constant and thus increase the range of propagation. Exact analytic expressions for the decay constant are obtained for the case of equally strong interactions and uniform as well as oscillating concentrations on the Bethe lattice. Our general findings are illustrated using a real network of protein-protein interactions in baker's yeast with experimentally determined protein concentrations.

Growth of equilibrium structures built from a large number of distinct component types.

PubMed

Hedges, Lester O; Mannige, Ranjan V; Whitelam, Stephen

2014-09-14

We use simple analytic arguments and lattice-based computer simulations to study the growth of structures made from a large number of distinct component types. Components possess 'designed' interactions, chosen to stabilize an equilibrium target structure in which each component type has a defined spatial position, as well as 'undesigned' interactions that allow components to bind in a compositionally-disordered way. We find that high-fidelity growth of the equilibrium target structure can happen in the presence of substantial attractive undesigned interactions, as long as the energy scale of the set of designed interactions is chosen appropriately. This observation may help explain why equilibrium DNA 'brick' structures self-assemble even if undesigned interactions are not suppressed [Ke et al. Science, 338, 1177, (2012)]. We also find that high-fidelity growth of the target structure is most probable when designed interactions are drawn from a distribution that is as narrow as possible. We use this result to suggest how to choose complementary DNA sequences in order to maximize the fidelity of multicomponent self-assembly mediated by DNA. We also comment on the prospect of growing macroscopic structures in this manner.

A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

PubMed

Morris, Gavin; Fanucchi, Sylvia

2016-04-05

Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

PubMed

Binns, D D; Helms, M K; Barylko, B; Davis, C T; Jameson, D M; Albanesi, J P; Eccleston, J F

2000-06-20

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

The Free Energy Profile of Tubulin Straight-Bent Conformational Changes, with Implications for Microtubule Assembly and Drug Discovery

PubMed Central

Bonomi, Massimiliano; Agard, David A.; Jacobson, Matthew P.

2014-01-01

αβ-tubulin dimers need to convert between a ‘bent’ conformation observed for free dimers in solution and a ‘straight’ conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol) and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated. PMID:24516374

Cu(II) binding by a pH-fractionated fulvic acid

USGS Publications Warehouse

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

1999-01-01

The relationship between acidity, Cu(II) binding and sorption to XAD resin was examined using Suwannee River fulvic acid (SRFA). The work was based on the hypothesis that fractions of SRFA eluted from an XAD column at various pH's from 1.0 to 12.0 would show systematic variations in acidity and possibly aromaticity which in turn would lead to different Cu(II) binding properties. We measured equilibrium Cu(II) binding to these fractions using Cu2+ ion-selective electrode (ISE) potentiometry at pH 6.0. Several model ligands were also examined, including cyclopentane-1,2,3,4-tetracarboxylic acid (CP-TCA) and tetrahydrofuran-2,3,4,5-tetracarboxylic acid (THF-TCA), the latter binding Cu(II) much more strongly as a consequence of the ether linkage. The SRFA Cu(II) binding properties agreed with previous work at high ionic strength, and binding was enhanced substantially at lower ionic strength, in agreement with Poisson-Boltzmann predictions for small spheres. Determining Cu binding constants (K(i)) by non-linear regression with total ligand concentrations (L(Ti)) taken from previous work, the fractions eluted at varying pH had K(i) similar to the unfractionated SRFA, with a maximum enhancement of 0.50 log units. We conclude that variable-pH elution from XAD does not isolate significantly strong (or weak) Cu(II)-binding components from the SRFA mixture. Copyright (C) 1999 Elsevier Science B.V.

In vivo potency revisited - Keep the target in sight.

PubMed

Gabrielsson, Johan; Peletier, Lambertus A; Hjorth, Stephan

2018-04-01

Potency is a central parameter in pharmacological and biochemical sciences, as well as in drug discovery and development endeavors. It is however typically defined in terms only of ligand to target binding affinity also in in vivo experimentation, thus in a manner analogous to in in vitro studies. As in vivo potency is in fact a conglomerate of events involving ligand, target, and target-ligand complex processes, overlooking some of the fundamental differences between in vivo and in vitro may result in serious mispredictions of in vivo efficacious dose and exposure. The analysis presented in this paper compares potency measures derived from three model situations. Model A represents the closed in vitro system, defining target binding of a ligand when total target and ligand concentrations remain static and constant. Model B describes an open in vivo system with ligand input and clearance (Cl (L) ), adding in parallel to the turnover (k syn , k deg ) of the target. Model C further adds to the open in vivo system in Model B also the elimination of the target-ligand complex (k e(RL) ) via a first-order process. We formulate corresponding equations of the equilibrium (steady-state) relationships between target and ligand, and complex and ligand for each of the three model systems and graphically illustrate the resulting simulations. These equilibrium relationships demonstrate the relative impact of target and target-ligand complex turnover, and are easier to interpret than the more commonly used ligand-, target- and complex concentration-time courses. A new potency expression, labeled L 50 , is then derived. L 50 is the ligand concentration at half-maximal target and complex concentrations and is an amalgamation of target turnover, target-ligand binding and complex elimination parameters estimated from concentration-time data. L 50 is then compared to the dissociation constant K d (target-ligand binding affinity), the conventional Black & Leff potency estimate EC 50 , and the derived Michaelis-Menten parameter K m (target-ligand binding and complex removal) across a set of literature data. It is evident from a comparison between parameters derived from in vitro vs. in vivo experiments that L 50 can be either numerically greater or smaller than the K d (or K m ) parameter, primarily depending on the ratio of k deg -to-k e(RL) . Contrasting the limit values of target R and target-ligand complex RL for ligand concentrations approaching infinity demonstrates that the outcome of the three models differs to a great extent. Based on the analysis we propose that a better understanding of in vivo pharmacological potency requires simultaneous assessment of the impact of its underlying determinants in the open system setting. We propose that L 50 will be a useful parameter guiding predictions of the effective concentration range, for translational purposes, and assessment of in vivo target occupancy/suppression by ligand, since it also encompasses target turnover - in turn also subject to influence by pathophysiology and drug treatment. Different compounds may have similar binding affinity for a target in vitro (same K d ), but vastly different potencies in vivo. L 50 points to what parameters need to be taken into account, and particularly that closed-system (in vitro) parameters should not be first choice when ranking compounds in vivo (open system). Copyright © 2017 Elsevier Inc. All rights reserved.

Optical binding with cold atoms

NASA Astrophysics Data System (ADS)

Máximo, C. E.; Bachelard, R.; Kaiser, R.

2018-04-01

Optical binding is a form of light-mediated forces between elements of matter which emerge in response to the collective scattering of light. Such a phenomenon has been studied mainly in the context of the equilibrium stability of dielectric sphere arrays which move amid dissipative media. In this article, we demonstrate that optically bounded states of a pair of cold atoms can exist, in the absence of nonradiative damping. We study the scaling laws for the unstable-stable phase transition at negative detuning and the unstable-metastable one for positive detuning. In addition, we show that angular momentum can lead to dynamical stabilization with infinite-range scaling.

Gel Filtration Of Dilute Human Embryonic Hemoglobins Reveals Basis For Their Increased Oxygen Binding

PubMed Central

Manning, Lois R.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, James M.

2016-01-01

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. PMID:27965062

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding.

PubMed

Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C; Chait, Brian T; Manning, James M

2017-02-15

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. Copyright © 2016 Elsevier Inc. All rights reserved.

ENTRAPMENT OF PROTEINS IN GLYCOGEN-CAPPED AND HYDRAZIDE-ACTIVATED SUPPORTS

PubMed Central

Jackson, Abby J.; Xuan, Hai; Hage, David S.

2010-01-01

A method is described for the entrapment of proteins in hydrazide-activated supports using oxidized glycogen as a capping agent. This approach is demonstrated using human serum albumin (HSA) as a model binding agent. After optimization of this method, a protein content of 43 (± 1) mg HSA/g support was obtained for porous silica. The entrapped HSA supports could retain a low mass drug (S-warfarin) and had activities and equilibrium constants comparable to those for soluble HSA. It was also found that this approach could be used with other proteins and binding agents that had masses between 5.8 and 150 kDa. PMID:20470745

Sequential memory: Binding dynamics

NASA Astrophysics Data System (ADS)

Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

2015-10-01

Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

The binding of glucose to yeast hexokinase monomers is independent of ionic strength.

PubMed

Mayes, E L; Hoggett, J G; Kellett, G L

1982-05-01

Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer.

The binding of glucose to yeast hexokinase monomers is independent of ionic strength.

PubMed Central

Mayes, E L; Hoggett, J G; Kellett, G L

1982-01-01

Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer. PMID:7052060

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

PubMed Central

Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

2011-01-01

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

Sequential memory: Binding dynamics.

PubMed

Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

2015-10-01

Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories-episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Coherent Conformational Degrees of Freedom as a Structural Basis for Allosteric Communication

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites. PMID:22174669

CFD analysis of laboratory scale phase equilibrium cell operation

NASA Astrophysics Data System (ADS)

Jama, Mohamed Ali; Nikiforow, Kaj; Qureshi, Muhammad Saad; Alopaeus, Ville

2017-10-01

For the modeling of multiphase chemical reactors or separation processes, it is essential to predict accurately chemical equilibrium data, such as vapor-liquid or liquid-liquid equilibria [M. Šoóš et al., Chem. Eng. Process.: Process Intensif. 42(4), 273-284 (2003)]. The instruments used in these experiments are typically designed based on previous experiences, and their operation verified based on known equilibria of standard components. However, mass transfer limitations with different chemical systems may be very different, potentially falsifying the measured equilibrium compositions. In this work, computational fluid dynamics is utilized to design and analyze laboratory scale experimental gas-liquid equilibrium cell for the first time to augment the traditional analysis based on plug flow assumption. Two-phase dilutor cell, used for measuring limiting activity coefficients at infinite dilution, is used as a test case for the analysis. The Lagrangian discrete model is used to track each bubble and to study the residence time distribution of the carrier gas bubbles in the dilutor cell. This analysis is necessary to assess whether the gas leaving the cell is in equilibrium with the liquid, as required in traditional analysis of such apparatus. Mass transfer for six different bio-oil compounds is calculated to determine the approach equilibrium concentration. Also, residence times assuming plug flow and ideal mixing are used as reference cases to evaluate the influence of mixing on the approach to equilibrium in the dilutor. Results show that the model can be used to predict the dilutor operating conditions for which each of the studied gas-liquid systems reaches equilibrium.

CFD analysis of laboratory scale phase equilibrium cell operation.

PubMed

Jama, Mohamed Ali; Nikiforow, Kaj; Qureshi, Muhammad Saad; Alopaeus, Ville

2017-10-01

For the modeling of multiphase chemical reactors or separation processes, it is essential to predict accurately chemical equilibrium data, such as vapor-liquid or liquid-liquid equilibria [M. Šoóš et al., Chem. Eng. Process Intensif. 42(4), 273-284 (2003)]. The instruments used in these experiments are typically designed based on previous experiences, and their operation verified based on known equilibria of standard components. However, mass transfer limitations with different chemical systems may be very different, potentially falsifying the measured equilibrium compositions. In this work, computational fluid dynamics is utilized to design and analyze laboratory scale experimental gas-liquid equilibrium cell for the first time to augment the traditional analysis based on plug flow assumption. Two-phase dilutor cell, used for measuring limiting activity coefficients at infinite dilution, is used as a test case for the analysis. The Lagrangian discrete model is used to track each bubble and to study the residence time distribution of the carrier gas bubbles in the dilutor cell. This analysis is necessary to assess whether the gas leaving the cell is in equilibrium with the liquid, as required in traditional analysis of such apparatus. Mass transfer for six different bio-oil compounds is calculated to determine the approach equilibrium concentration. Also, residence times assuming plug flow and ideal mixing are used as reference cases to evaluate the influence of mixing on the approach to equilibrium in the dilutor. Results show that the model can be used to predict the dilutor operating conditions for which each of the studied gas-liquid systems reaches equilibrium.

Electrostatic forces govern the binding mechanism of intrinsically disordered histone chaperones

PubMed Central

Liu, Chuanbo; Wang, Tianshu; Bai, Yawen; Wang, Jin

2017-01-01

A unified picture to understand the protein recognition and function must include the native binding complex structure ensembles and the underlying binding mechanisms involved in specific biological processes. However, quantifications of both binding complex structures and dynamical mechanisms are still challenging for IDP. In this study, we have investigated the underlying molecular mechanism of the chaperone Chz1 and histone H2A.Z-H2B association by equilibrium and kinetic stopped-flow fluorescence spectroscopy. The dependence of free energy and kinetic rate constant on electrolyte mean activity coefficient and urea concentration are uncovered. Our results indicate a previous unseen binding kinetic intermediate. An initial conformation selection step of Chz1 is also revealed before the formation of this intermediate state. Based on these observations, a mixed mechanism of three steps including both conformation selection and induced fit is proposed. By combination of the ion- and denaturant-induced experiments, we demonstrate that electrostatic forces play a dominant role in the recognition of bipolar charged intrinsically disordered protein Chz1 to its preferred partner H2A.Z-H2B. Both the intra-chain and inter-chain electrostatic interactions have direct impacts on the native collapsed structure and binding mechanism. PMID:28552960

Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.

1985-02-01

A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using (/sup 3/H)scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer withmore » the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.« less

Spectroscopic and microcalorimetric studies on the molecular binding of food colorant acid red 27 with deoxyribonucleic acid.

PubMed

Basu, Anirban; Kumar, Gopinatha Suresh

2016-08-01

Interaction of the food colorant acid red 27 with double stranded DNA was investigated using spectroscopic and calorimetric methods. Absorbance and fluorescence studies suggested an intimate binding interaction between the dye and DNA. The quantum efficiency value testified an effective energy transfer from the DNA base pairs to the dye molecules. Minor groove displacement assay with Hoechst 33258 revealed that the binding occurs in the minor groove of DNA. Circular dichroism studies revealed that acid red 27 induces moderate conformational perturbations in DNA. Results of calorimetric studies suggested that the complexation process was driven largely by positive entropic contribution with a smaller favorable enthalpy contribution. The equilibrium constant of the binding was calculated to be (3.04 ± 0.09) × 10(4)  M(-1) at 298.15 K. Negative heat capacity value along with the enthalpy-entropy compensation phenomenon established the involvement of dominant hydrophobic forces in the binding process. Differential scanning calorimetry studies presented evidence for an increased thermal stability of DNA on binding of acid red 27. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

PubMed Central

Hytönen, Vesa P; Vogel, Viola

2008-01-01

Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

PubMed Central

James, Kevin A.; Verkhivker, Gennady M.

2014-01-01

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues. PMID:25427151

Structure and binding energy of the H2S dimer at the CCSD(T) complete basis set limit.

PubMed

Lemke, Kono H

2017-06-21

This study presents results for the binding energy and geometry of the H 2 S dimer which have been computed using Møller-Plesset perturbation theory (MP2, MP4) and coupled cluster (CCSD, CCSD(T)) calculations with basis sets up to aug-cc-pV5Z. Estimates of D e , E ZPE , D o , and dimer geometry have been obtained at each level of theory by taking advantage of the systematic convergence behavior toward the complete basis set (CBS) limit. The CBS limit binding energy values of D e are 1.91 (MP2), 1.75 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD[T]). The most accurate values for the equilibrium S-S distance r SS (without counterpoise correction) are 4.080 (MP2/aug-cc-pV5Z), 4.131 (MP4/aug-cc-pVQZ), 4.225 (CCSD/aug-cc-pVQZ), and 4.146 Å (CCSD(T)/aug-cc-pVQZ). This study also evaluates the effect of counterpoise correction on the H 2 S dimer geometry and binding energy. As regards the structure of (H 2 S) 2 , MPn, CCSD, and CCSD(T) level values of r SS , obtained by performing geometry optimizations on the counterpoise-corrected potential energy surface, converge systematically to CBS limit values of 4.099 (MP2), 4.146 (MP4), 4.233 (CCSD), and 4.167 Å (CCSD(T)). The corresponding CBS limit values of the equilibrium binding energy D e are 1.88 (MP2), 1.76 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD(T)), the latter in excellent agreement with the measured binding energy value of 1.68 ± 0.02 kcal/mol reported by Ciaffoni et al. [Appl. Phys. B 92, 627 (2008)]. Combining CBS electronic binding energies D e with E ZPE predicted by CCSD(T) vibrational second-order perturbation theory calculations yields D o = 1.08 kcal/mol, which is around 0.6 kcal/mol smaller than the measured value of 1.7 ± 0.3 kcal/mol. Overall, the results presented here demonstrate that the application of high level calculations, in particular CCSD(T), in combination with augmented correlation consistent basis sets provides valuable insight into the structure and energetics of the hydrogen sulfide dimer.

Structure and binding energy of the H2S dimer at the CCSD(T) complete basis set limit

NASA Astrophysics Data System (ADS)

Lemke, Kono H.

2017-06-01

This study presents results for the binding energy and geometry of the H2S dimer which have been computed using Møller-Plesset perturbation theory (MP2, MP4) and coupled cluster (CCSD, CCSD(T)) calculations with basis sets up to aug-cc-pV5Z. Estimates of De, EZPE, Do, and dimer geometry have been obtained at each level of theory by taking advantage of the systematic convergence behavior toward the complete basis set (CBS) limit. The CBS limit binding energy values of De are 1.91 (MP2), 1.75 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD[T]). The most accurate values for the equilibrium S-S distance rSS (without counterpoise correction) are 4.080 (MP2/aug-cc-pV5Z), 4.131 (MP4/aug-cc-pVQZ), 4.225 (CCSD/aug-cc-pVQZ), and 4.146 Å (CCSD(T)/aug-cc-pVQZ). This study also evaluates the effect of counterpoise correction on the H2S dimer geometry and binding energy. As regards the structure of (H2S)2, MPn, CCSD, and CCSD(T) level values of rSS, obtained by performing geometry optimizations on the counterpoise-corrected potential energy surface, converge systematically to CBS limit values of 4.099 (MP2), 4.146 (MP4), 4.233 (CCSD), and 4.167 Å (CCSD(T)). The corresponding CBS limit values of the equilibrium binding energy De are 1.88 (MP2), 1.76 (MP4), 1.41 (CCSD), and 1.69 kcal/mol (CCSD(T)), the latter in excellent agreement with the measured binding energy value of 1.68 ± 0.02 kcal/mol reported by Ciaffoni et al. [Appl. Phys. B 92, 627 (2008)]. Combining CBS electronic binding energies De with EZPE predicted by CCSD(T) vibrational second-order perturbation theory calculations yields Do = 1.08 kcal/mol, which is around 0.6 kcal/mol smaller than the measured value of 1.7 ± 0.3 kcal/mol. Overall, the results presented here demonstrate that the application of high level calculations, in particular CCSD(T), in combination with augmented correlation consistent basis sets provides valuable insight into the structure and energetics of the hydrogen sulfide dimer.

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops. Copyright 2001 Academic Press.

Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.

PubMed

Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira

2011-08-01

Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).

Cato Guldberg and Peter Waage, the history of the Law of Mass Action, and its relevance to clinical pharmacology.

PubMed

Ferner, Robin E; Aronson, Jeffrey K

2016-01-01

We have traced the historical link between the Law of Mass Action and clinical pharmacology. The Law evolved from the work of the French chemist Claude Louis Berthollet, was first formulated by Cato Guldberg and Peter Waage in 1864 and later clarified by the Dutch chemist Jacobus van 't Hoff in 1877. It has profoundly influenced our qualitative and quantitative understanding of a number of physiological and pharmacological phenomena. According to the Law of Mass Action, the velocity of a chemical reaction depends on the concentrations of the reactants. At equilibrium the concentrations of the chemicals involved bear a constant relation to each other, described by the equilibrium constant, K. The Law of Mass Action is relevant to various physiological and pharmacological concepts, including concentration-effect curves, dose-response curves, and ligand-receptor binding curves, all of which are important in describing the pharmacological actions of medications, the Langmuir adsorption isotherm, which describes the binding of medications to proteins, activation curves for transmembrane ion transport, enzyme inhibition and the Henderson-Hasselbalch equation, which describes the relation between pH, as a measure of acidity and the concentrations of the contributory acids and bases. Guldberg and Waage recognized the importance of dynamic equilibrium, while others failed to do so. Their ideas, over 150 years old, are embedded in and still relevant to clinical pharmacology. Here we explain the ideas and in a subsequent paper show how they are relevant to understanding adverse drug reactions. © 2015 The British Pharmacological Society.

Mathematical simulations for bioanalytical assay development: the (un-)necessity and (im-)possibility of free drug quantification.

PubMed

Staack, Roland F; Jordan, Gregor; Heinrich, Julia

2012-02-01

For every drug development program it needs to be discussed whether discrimination between free and total drug concentrations is required to accurately describe its pharmacokinetic behavior. This perspective describes the application of mathematical simulation approaches to guide this initial decision based on available knowledge about target biology, binding kinetics and expected drug concentrations. We provide generic calculations that can be used to estimate the necessity of free drug quantification for different drug molecules. In addition, mathematical approaches are used to simulate various assay conditions in bioanalytical ligand-binding assays: it is demonstrated that due to the noncovalent interaction between the binding partners and typical assay-related interferences in the equilibrium, a correct quantification of the free drug concentration is highly challenging and requires careful design of different assay procedure steps.

Intermolecular symmetry-adapted perturbation theory study of large organic complexes.

PubMed

Heßelmann, Andreas; Korona, Tatiana

2014-09-07

Binding energies for the complexes of the S12L database by Grimme [Chem. Eur. J. 18, 9955 (2012)] were calculated using intermolecular symmetry-adapted perturbation theory combined with a density-functional theory description of the interacting molecules. The individual interaction energy decompositions revealed no particular change in the stabilisation pattern as compared to smaller dimer systems at equilibrium structures. This demonstrates that, to some extent, the qualitative description of the interaction of small dimer systems may be extrapolated to larger systems, a method that is widely used in force-fields in which the total interaction energy is decomposed into atom-atom contributions. A comparison of the binding energies with accurate experimental reference values from Grimme, the latter including thermodynamic corrections from semiempirical calculations, has shown a fairly good agreement to within the error range of the reference binding energies.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-02-22

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

Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete†

PubMed Central

Tjepkema, John D.; Cashon, Robert E.; Beckwith, Jason; Schwintzer, Christa R.

2002-01-01

Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1. PMID:11976149

Tautomerism, Hammett σ, and QSAR

NASA Astrophysics Data System (ADS)

Martin, Yvonne Connolly

2010-06-01

A consideration of equilibrium model-based equations suggests that tautomeric equilibria do not markedly affect observed potency if the tautomer bound represents at least 50% of the compound in solution. Tautomeric equilibria can enhance or attenuate the correlation of potency with Hammett σ. Additionally, tautomeric equilibria can lead to a correlation of potency with σ even in the absence of a correlation of binding with σ.

MODEL FOR NON-EQUILIBRIUM BINDING AND AFFINITY CHROMATOGRAPHY WITH CHARACTERIZATION OF 8-HYDROXYQUINOLINE IMMOBILIZED ON CONTROLLED PORE GLASS USING A FLOW INJECTION SYSTEM WITH A PACKED MICRO-COLUMN. (R826694C651)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Report of a Workshop on Membrane Biophysics Held June 25 - July 15, 1984.

DTIC Science & Technology

1984-07-15

J.A. (1984) Identification of aggregated Markovian models: application to the nicotinic acetylcholine receptor. Prof. of the Neyman-Kiefer Memorial...for future biological applications of the system. We have also discovered that the kinetic and equilibrium 0 properties of the binding depend...consists of aggregation of phycobiliproteins, -- namely phycoerythrin, phycocyanin , and allophycocyanin, which collectively form the phycobilisomes. Each

Ab Initio Studies of Shock-Induced Chemical Reactions of Inter-Metallics

NASA Astrophysics Data System (ADS)

Zaharieva, Roussislava; Hanagud, Sathya

2009-06-01

Shock-induced and shock assisted chemical reactions of intermetallic mixtures are studied by many researchers, using both experimental and theoretical techniques. The theoretical studies are primarily at continuum scales. The model frameworks include mixture theories and meso-scale models of grains of porous mixtures. The reaction models vary from equilibrium thermodynamic model to several non-equilibrium thermodynamic models. The shock-effects are primarily studied using appropriate conservation equations and numerical techniques to integrate the equations. All these models require material constants from experiments and estimates of transition states. Thus, the objective of this paper is to present studies based on ab initio techniques. The ab inito studies, to date, use ab inito molecular dynamics. This paper presents a study that uses shock pressures, and associated temperatures as starting variables. Then intermetallic mixtures are modeled as slabs. The required shock stresses are created by straining the lattice. Then, ab initio binding energy calculations are used to examine the stability of the reactions. Binding energies are obtained for different strain components super imposed on uniform compression and finite temperatures. Then, vibrational frequencies and nudge elastic band techniques are used to study reactivity and transition states. Examples include Ni and Al.

Zinc sorption to three gram-negative bacteria: combined titration, modeling, and EXAFS study.

PubMed

Guiné, V; Spadini, L; Sarret, G; Muris, M; Delolme, C; Gaudet, J P; Martins, J M F

2006-03-15

The acid-base and Zn sorption properties of three bacteria, Cupriavidus metallidurans CH34, Pseudomonas putida ATCC12633, and Escherichia coli K12DH5alpha, were investigated through an original combination of extended X-ray absorption fine structure (EXAFS) spectroscopy and equilibrium titration studies. Acid-base titration curves of the three strains were fitted with a model accounting for three conceptual reactive sites: an acidic (carboxyl and/or phosphodiester), a neutral (phosphomonoester), and a basic (amine and/or hydroxyl) group. Calculated proton and Zn equilibrium constants and site densities compare with literature data. The nature of Zn binding sites was studied by EXAFS spectroscopy. Phosphoester, carboxyl, and unexpectedly sulfhydryl ligands were identified. Their proportions depended on Zn loading and bacterial strain and were consistent with the titration results. These findings were compared to the structure and site density of the major cell wall components. It appeared that the cumulated theoretical site density of these structures (<2 Zn nm(-2)) was much lower than the total site density of the investigated strains (16-56 Zn nm(-2)). These results suggest a dominant role of extracellular polymeric substances in Zn retention processes, although Zn binding to inner cell components cannot be excluded.

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

PubMed

Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

2011-11-01

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

A general intermolecular force field based on tight-binding quantum chemical calculations

NASA Astrophysics Data System (ADS)

Grimme, Stefan; Bannwarth, Christoph; Caldeweyher, Eike; Pisarek, Jana; Hansen, Andreas

2017-10-01

A black-box type procedure is presented for the generation of a molecule-specific, intermolecular potential energy function. The method uses quantum chemical (QC) information from our recently published extended tight-binding semi-empirical scheme (GFN-xTB) and can treat non-covalently bound complexes and aggregates with almost arbitrary chemical structure. The necessary QC information consists of the equilibrium structure, Mulliken atomic charges, charge centers of localized molecular orbitals, and also of frontier orbitals and orbital energies. The molecular pair potential includes model density dependent Pauli repulsion, penetration, as well as point charge electrostatics, the newly developed D4 dispersion energy model, Drude oscillators for polarization, and a charge-transfer term. Only one element-specific and about 20 global empirical parameters are needed to cover systems with nuclear charges up to radon (Z = 86). The method is tested for standard small molecule interaction energy benchmark sets where it provides accurate intermolecular energies and equilibrium distances. Examples for structures with a few hundred atoms including charged systems demonstrate the versatility of the approach. The method is implemented in a stand-alone computer code which enables rigid-body, global minimum energy searches for molecular aggregation or alignment.

Calcium-controlled conformational choreography in the N-terminal half of adseverin

NASA Astrophysics Data System (ADS)

Chumnarnsilpa, Sakesit; Robinson, Robert C.; Grimes, Jonathan M.; Leyrat, Cedric

2015-09-01

Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1-A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1-A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1-A3, albeit at a lower efficiency than observed for gelsolin domains G1-G3. Together, these data address the calcium dependency of A1-A3 activity in relation to the calcium-independent activity of G1-G3.

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

PubMed Central

Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP3 on plasma membrane-like bilayers reveals that dimeric WT PH domain possesses a one-order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically-enhanced affinity for one PIP3 molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP3 molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP3 affinity and bound state lifetime that are each a full order of magnitude lower than dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer-monomer conversion. PMID:23745598

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

PubMed

Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows deeper insertion of the protein into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP3 on plasma membrane-like bilayers reveal that the dimeric WT PH domain possesses a one order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically enhanced affinity for one PIP3 molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP3 molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP3 affinity and bound state lifetime that are each 1 order of magnitude lower than those of the dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to the cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer-monomer conversion.

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

PubMed

Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

2009-01-01

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta

DOE PAGES

Jiang, Lin; Liu, Cong; Leibly, David; ...

2013-07-16

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind tomore » Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.« less

Noninvasive bi-graphical analysis for the quantification of slowly reversible radioligand binding

NASA Astrophysics Data System (ADS)

Seo, Seongho; Kim, Su Jin; Yoo, Hye Bin; Lee, Jee-Young; Kyeong Kim, Yu; Lee, Dong Soo; Zhou, Yun; Lee, Jae Sung

2016-09-01

In this paper, we presented a novel reference-region-based (noninvasive) bi-graphical analysis for the quantification of a reversible radiotracer binding that may be too slow to reach relative equilibrium (RE) state during positron emission tomography (PET) scans. The proposed method indirectly implements the noninvasive Logan plot, through arithmetic combination of the parameters of two other noninvasive methods and the apparent tissue-to-plasma efflux rate constant for the reference region (k2\\prime ). We investigated its validity and statistical properties, by performing a simulation study with various noise levels and k2\\prime values, and also evaluated its feasibility for [18F]FP-CIT PET in human brain. The results revealed that the proposed approach provides distribution volume ratio estimation comparable to the Logan plot at low noise levels while improving underestimation caused by non-RE state differently depending on k2\\prime . Furthermore, the proposed method was able to avoid noise-induced bias of the Logan plot, and the variability of its results was less dependent on k2\\prime than the Logan plot. Therefore, this approach, without issues related to arterial blood sampling given a pre-estimate of k2\\prime (e.g. population-based), could be useful in parametric image generation for slow kinetic tracers staying in a non-RE state within a PET scan.

Stereoselective binding of doxazosin enantiomers to plasma proteins from rats, dogs and humans in vitro

PubMed Central

Sun, Jia-an; Kong, De-zhi; Zhen, Ya-qin; Li, Qing; Zhang, Wei; Zhang, Jiang-hua; Yin, Zhi-wei; Ren, Lei-ming

2013-01-01

Aim: (±)Doxazosin is a long-lasting inhibitor of α1-adrenoceptors that is widely used to treat benign prostatic hyperplasia and lower urinary tract symptoms. In this study we investigated the stereoselective binding of doxazosin enantiomers to the plasma proteins of rats, dogs and humans in vitro. Methods: Human, dog and rat plasma were prepared. Equilibrium dialysis was used to determine the plasma protein binding of each enantiomer in vitro. Chiral HPLC with fluorescence detection was used to measure the drug concentrations on each side of the dialysis membrane bag. Results: Both the enantiomers were highly bound to the plasma proteins of rats, dogs and humans [(−)doxazosin: 89.4%–94.3%; (+)doxazosin: 90.9%–95.4%]. (+)Doxazosin exhibited significantly higher protein binding capacities than (−)doxazosin in all the three species, and the difference in the bound concentration (Cb) between the two enantiomers was enhanced as their concentrations were increased. Although the percentage of the plasma protein binding in the dog plasma was significantly lower than that in the human plasma at 400 and 800 ng/mL, the corrected percentage of plasma protein binding was dog>human>rat. Conclusion: (−)Doxazosin and (+)doxazosin show stereoselective plasma protein binding with a significant species difference among rats, dogs and humans. PMID:24241343

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

PubMed

Torres, Oscar B; Duval, Alexander J; Sulima, Agnieszka; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

2018-06-01

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Energy transfer between two vacuum-gapped metal plates: Coulomb fluctuations and electron tunneling

NASA Astrophysics Data System (ADS)

Zhang, Zu-Quan; Lü, Jing-Tao; Wang, Jian-Sheng

2018-05-01

Recent experimental measurements for near-field radiative heat transfer between two bodies have been able to approach the gap distance within 2 nm , where the contributions of Coulomb fluctuation and electron tunneling are comparable. Using the nonequilibrium Green's function method in the G0W0 approximation, based on a tight-binding model, we obtain for the energy current a Caroli formula from the Meir-Wingreen formula in the local equilibrium approximation. Also, the Caroli formula is consistent with the evanescent part of the heat transfer from the theory of fluctuational electrodynamics. We go beyond the local equilibrium approximation to study the energy transfer in the crossover region from electron tunneling to Coulomb fluctuation based on a numerical calculation.

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

PubMed

Shimazaki, K; Sato, M; Takegami, T

1981-11-05

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

Preparation of ellagic acid molecularly imprinted polymeric microspheres based on distillation-precipitation polymerization for the efficient purification of a crude extract.

PubMed

Zhang, Hua; Zhao, Shangge; Zhang, Lu; Han, Bo; Yao, Xincheng; Chen, Wen; Hu, Yanli

2016-08-01

Molecularly imprinted polymeric microspheres with a high recognition ability toward the template molecule, ellagic acid, were synthesized based on distillation-precipitation polymerization. The as-obtained polymers were characterized by scanning electron microscopy, infrared spectroscopy, and thermogravimetric analysis. Static, dynamic, and selective binding tests were adopted to study the binding properties and the molecular recognition ability of the prepared polymers for ellagic acid. The results indicated that the maximum static adsorption capacity of the prepared polymers toward ellagic acid was 37.07 mg/g and the adsorption equilibrium time was about 100 min when the concentration of ellagic acid was 40 mg/mL. Molecularly imprinted polymeric microspheres were also highly selective toward ellagic acid compared with its analogue quercetin. It was found that the content of ellagic acid in the pomegranate peel extract was enhanced from 23 to 86% after such molecularly imprinted solid-phase extraction process. This work provides an efficient way for effective separation and enrichment of ellagic acid from complex matrix, which is especially valuable in industrial production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subnanosecond spectrofluorimetry of new indolocarbazole derivatives in solutions and protein complexes and their dipole moments

NASA Astrophysics Data System (ADS)

Nemkovich, N. A.; Kruchenok, Yu. V.; Sobchuk, A. N.; Detert, H.; Wrobel, N.; Chernyavskiĭ, E. A.

2009-08-01

The spectral and temporal characteristics of new 6,12-dimethoxyindolo[3,2- b]carbazole, 5,11-dimethyl-6,12-dimethoxyindolo[3,2- b]carbazole, and 5,11-dihexyl-6,12-di(hexyloxy)indolo[3,2- b]carbazole fluorescence probes in organic solvents and protein complexes are studied. The dipole moments of indolocarbazoles in 1,4-dioxane were measured by electrooptical absorption method. The measured dipole moments have values within the range of (3.1-3.6) × 10-30 C m in the equilibrium ground state and increase to (4.8-5.6) × 10-30 C m after excitation. The excited state lifetime of indolocarbazole derivatives increases with increasing polarity and viscosity of the environment. The binding of indolocarbazoles with trypsinogen and human serum albumin increases the fluorescence intensity, changes the intensity ratio of fluorescence bands, and increases the average excited state lifetime of indolocarbazoles. The analysis of the instantaneous fluorescence spectra and fluorescence decay parameters at different wavelengths revealed the existence of several types of probe binding sites in proteins. It is found that the fluorescence characteristics of indolocarbazole derivatives depend on the conformation rearrangements of trypsinogen due to its thermal denaturation.

A framework for modelling gene regulation which accommodates non-equilibrium mechanisms.

PubMed

Ahsendorf, Tobias; Wong, Felix; Eils, Roland; Gunawardena, Jeremy

2014-12-05

Gene regulation has, for the most part, been quantitatively analysed by assuming that regulatory mechanisms operate at thermodynamic equilibrium. This formalism was originally developed to analyse the binding and unbinding of transcription factors from naked DNA in eubacteria. Although widely used, it has made it difficult to understand the role of energy-dissipating, epigenetic mechanisms, such as DNA methylation, nucleosome remodelling and post-translational modification of histones and co-regulators, which act together with transcription factors to regulate gene expression in eukaryotes. Here, we introduce a graph-based framework that can accommodate non-equilibrium mechanisms. A gene-regulatory system is described as a graph, which specifies the DNA microstates (vertices), the transitions between microstates (edges) and the transition rates (edge labels). The graph yields a stochastic master equation for how microstate probabilities change over time. We show that this framework has broad scope by providing new insights into three very different ad hoc models, of steroid-hormone responsive genes, of inherently bounded chromatin domains and of the yeast PHO5 gene. We find, moreover, surprising complexity in the regulation of PHO5, which has not yet been experimentally explored, and we show that this complexity is an inherent feature of being away from equilibrium. At equilibrium, microstate probabilities do not depend on how a microstate is reached but, away from equilibrium, each path to a microstate can contribute to its steady-state probability. Systems that are far from equilibrium thereby become dependent on history and the resulting complexity is a fundamental challenge. To begin addressing this, we introduce a graph-based concept of independence, which can be applied to sub-systems that are far from equilibrium, and prove that history-dependent complexity can be circumvented when sub-systems operate independently. As epigenomic data become increasingly available, we anticipate that gene function will come to be represented by graphs, as gene structure has been represented by sequences, and that the methods introduced here will provide a broader foundation for understanding how genes work.

Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein.

PubMed

Damberger, Fred F; Michel, Erich; Ishida, Yuko; Leal, Walter S; Wüthrich, Kurt

2013-11-12

The Bombyx mori pheromone-binding protein (BmorPBP) is known to adopt two different conformations. These are BmorPBP(A), where a regular helix formed by the C-terminal dodecapeptide segment, α7, occupies the ligand-binding cavity, and BmorPBP(B), where the binding site is free to accept ligands. NMR spectra of delipidated BmorPBP solutions at the physiological pH of the bulk sensillum lymph near pH 6.5 show only BmorPBP(A), and in mixtures, the two species are in slow exchange on the chemical shift frequency scale. This equilibrium has been monitored at variable pH and ligand concentrations, demonstrating that it is an intrinsic property of BmorPBP that is strongly affected by pH variation and ligand binding. This polymorphism tunes BmorPBP for optimal selective pheromone transport: Competition between α7 and lipophilic ligands for its binding cavity enables selective uptake of bombykol at the pore endings in the sensillum wall, whereas compounds with lower binding affinity can only be bound in the bulk sensillum lymph. After transport across the bulk sensillum lymph into the lower pH area near the dendritic membrane surface, bombykol is ejected near the receptor, whereas compounds with lower binding affinity are ejected before reaching the olfactory receptor, rendering them susceptible to degradation by enzymes present in the sensillum lymph.

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

PubMed

Athwal, G S; Lombardo, C R; Huber, J L; Masters, S C; Fu, H; Huber, S C

2000-04-01

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.