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Anthony Mittermaier - One of the best experts on this subject based on the ideXlab platform.

  • rapid measurement of inhibitor binding kinetics by Isothermal Titration Calorimetry
    Nature Communications, 2018
    Co-Authors: Justin M Di Trani, Stephane De Cesco, Rebecca Oleary, Jessica Plescia, Claudia Jorge Do Nascimento, Nicolas Moitessier, Anthony Mittermaier
    Abstract:

    Although drug development typically focuses on binding thermodynamics, recent studies suggest that kinetic properties can strongly impact a drug candidate's efficacy. Robust techniques for measuring inhibitor association and dissociation rates are therefore essential. To address this need, we have developed a pair of complementary Isothermal Titration Calorimetry (ITC) techniques for measuring the kinetics of enzyme inhibition. The advantages of ITC over standard techniques include speed, generality, and versatility; ITC also measures the rate of catalysis directly, making it ideal for quantifying rapid, inhibitor-dependent changes in enzyme activity. We used our methods to study the reversible covalent and non-covalent inhibitors of prolyl oligopeptidase (POP). We extracted kinetics spanning three orders of magnitude, including those too rapid for standard methods, and measured sub-nM binding affinities below the typical ITC limit. These results shed light on the inhibition of POP and demonstrate the general utility of ITC-based enzyme inhibition kinetic measurements.

  • measuring rapid time scale reaction kinetics using Isothermal Titration Calorimetry
    Analytical Chemistry, 2017
    Co-Authors: Justin M Di Trani, Nicolas Moitessier, Anthony Mittermaier
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological interactions including molecular recognition and enzymatic catalysis. ITC-based kinetics measurements typically focus on reactions taking place over long time scales (tens of minutes or hours) in order to avoid complications due to the finite length of time needed detect heat flow in the calorimeter cell. While progress has been made toward analyzing more rapid reaction kinetics by ITC, the capabilities and limitations of this approach have not been thoroughly tested to date. Here, we report that the time resolution of commercial instruments is on the order of 0.2 s or less. We successfully performed rapid ITC kinetics assays with durations of just tens of seconds using the enzyme trypsin. This is substantially shorter than previous ITC enzyme measurements. However, we noticed that for short reaction durations, standard assumptions regarding the ITC instrument response led to signific...

  • Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry
    2017
    Co-Authors: Justin M. Di Trani, Nicolas Moitessier, Anthony Mittermaier
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful tool for acquiring both thermodynamic and kinetic data for biological interactions including molecular recognition and enzymatic catalysis. ITC-based kinetics measurements typically focus on reactions taking place over long time scales (tens of minutes or hours) in order to avoid complications due to the finite length of time needed detect heat flow in the calorimeter cell. While progress has been made toward analyzing more rapid reaction kinetics by ITC, the capabilities and limitations of this approach have not been thoroughly tested to date. Here, we report that the time resolution of commercial instruments is on the order of 0.2 s or less. We successfully performed rapid ITC kinetics assays with durations of just tens of seconds using the enzyme trypsin. This is substantially shorter than previous ITC enzyme measurements. However, we noticed that for short reaction durations, standard assumptions regarding the ITC instrument response led to significant deviations between calculated and measured ITC peak shapes. To address this issue, we developed an ITC empirical response model (ITC-ERM) that quantitatively reproduces ITC peak shapes for all reaction durations. Applying the ITC-ERM approach to another enzyme (prolyl oligopeptidase), we unexpectedly discovered non-Michaelis–Menten kinetics in short time-scale measurements that are absent in more typical long time-scale experiments and are obscured in short time-scale experiments when standard assumptions regarding the instrument response are made. This highlights the potential of ITC measurements of rapid time scale kinetics in conjunction with the ITC-ERM approach to shed new light on biological dynamics

Peter Faller - One of the best experts on this subject based on the ideXlab platform.

  • thermodynamic study of cu2 binding to the dahk and ghk peptides by Isothermal Titration Calorimetry itc with the weaker competitor glycine
    Journal of Biological Inorganic Chemistry, 2012
    Co-Authors: Ana Trapaidze, Christelle Hureau, Wojciech Bal, Mathias Winterhalter, Peter Faller
    Abstract:

    The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodynamic technique Isothermal Titration Calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10−14 M and 7.0 ± 1.0 × 10−14 M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e., corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodynamic parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in Isothermal Titration Calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately determined without the use of a competitor ligand.

  • zinc binding to amyloid β Isothermal Titration Calorimetry and zn competition experiments with zn sensors
    Biochemistry, 2007
    Co-Authors: Christine Talmard, And Anais Bouzan, Peter Faller
    Abstract:

    Aggregation of the peptide amyloid-β (Aβ) to amyloid plaques is a key event in Alzheimer's disease. According to the amyloid cascade hypothesis, Aβ aggregates are toxic to neurons via the production of reactive oxygen species and are hence directly involved in the cause of the disease. Zinc ions play an important role, because they are able to bind to Aβ and influence the aggregation properties. In the present work Isothermal Titration Calorimetry and Zn sensors (zincon, Newport Green, and zinquin) were used to investigate the interaction of Zn with the full-length Aβ1−40 and Aβ1−42, as well as the truncated Aβ1−16 and Aβ1−28. The results suggest that Zn binding to Aβ induces a release of ∼0.9 proton by the peptide. This correspond to the expected value upon Zn binding to the three histidines and indicates that further ligands are not deprotonated upon Zn binding. Such behavior is expected for carboxylates, but not the N-terminus. Moreover, the apparent dissociation constant (Kd,app) of Zn binding to all ...

Sandro Keller - One of the best experts on this subject based on the ideXlab platform.

  • integration and global analysis of Isothermal Titration Calorimetry data for studying macromolecular interactions
    Nature Protocols, 2016
    Co-Authors: Chad A Brautigam, Carolyn Vargas, Sandro Keller, Huaying Zhao, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a Titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single Titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric Titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.

  • Integration and global analysis of Isothermal Titration Calorimetry data for studying macromolecular interactions
    Nature protocols, 2016
    Co-Authors: Chad A Brautigam, Carolyn Vargas, Sandro Keller, Huaying Zhao, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a Titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single Titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric Titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.

  • high precision Isothermal Titration Calorimetry with automated peak shape analysis
    Analytical Chemistry, 2012
    Co-Authors: Sandro Keller, Carolyn Vargas, Chad A Brautigam, Huaying Zhao, Grzegorz Piszczek, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noi...

  • Revisiting the optimal c value for Isothermal Titration Calorimetry
    Analytical biochemistry, 2011
    Co-Authors: Jana Broecker, Carolyn Vargas, Sandro Keller
    Abstract:

    The precision with which the dissociation constant, KD, can be obtained from Isothermal Titration Calorimetry depends on, among other factors, the concentrations of the interacting species. The so-called c value—the ratio of analyte concentration to KD—should fall in the range of 1 to 1000 for reliable KD determination. On the basis of simulated, noise-free data, Biswas and Tsodikov [5] recently suggested an optimal c value of 5 to 20. By contrast, we find an optimum at c > 40 on determining the KD confidence intervals through simulations containing noise levels typical of state-of-the-art microcalorimeters.

  • monitoring detergent mediated solubilization and reconstitution of lipid membranes by Isothermal Titration Calorimetry
    Nature Protocols, 2009
    Co-Authors: Heiko Heerklotz, Alekos Tsamaloukas, Sandro Keller
    Abstract:

    Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by Isothermal Titration Calorimetry

Ilian Jelesarov - One of the best experts on this subject based on the ideXlab platform.

  • survey of the year 2008 applications of Isothermal Titration Calorimetry
    Journal of Molecular Recognition, 2010
    Co-Authors: Robert J Falconer, Ilian Jelesarov, Anita Penkova, Brett M Collins
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a fast, accurate and label-free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes. Not only binding thermodynamics but also catalytic reactions, conformational rearrangements, changes in protonation and molecular dissociations can be readily quantified by performing only a small number of ITC experiments. In this review, we highlight some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs. As is tradition in these reviews we have not attempted a comprehensive analysis of all 500 papers using ITC, but emphasize those reports that particularly captured our interest and that included more thorough discussions we consider exemplify the power of the technique and might serve to inspire other users.

  • a survey of the year 2007 literature on applications of Isothermal Titration Calorimetry
    Journal of Molecular Recognition, 2008
    Co-Authors: Sasa Bjelic, Ilian Jelesarov
    Abstract:

    Elucidation of the energetic principles of binding affinity and specificity is a central task in many branches of current sciences: biology, medicine, pharmacology, chemistry, material sciences, etc. In biomedical research, integral approaches combining structural information with in-solution biophysical data have proved to be a powerful way toward understanding the physical basis of vital cellular phenomena. Isothermal Titration Calorimetry (ITC) is a valuable experimental tool facilitating quantification of the thermodynamic parameters that characterize recognition processes involving biomacromolecules. The method provides access to all relevant thermodynamic information by performing a few experiments. In particular, ITC experiments allow to by-pass tedious and (rarely precise) procedures aimed at determining the changes in enthalpy and entropy upon binding by van't Hoff analysis. Notwithstanding limitations, ITC has now the reputation of being the "gold standard" and ITC data are widely used to validate theoretical predictions of thermodynamic parameters, as well as to benchmark the results of novel binding assays. In this paper, we discuss several publications from 2007 reporting ITC results. The focus is on applications in biologically oriented fields. We do not intend a comprehensive coverage of all newly accumulated information. Rather, we emphasize work which has captured our attention with originality and far-reaching analysis, or else has provided ideas for expanding the potential of the method.

  • Isothermal Titration Calorimetry and differential scanning Calorimetry as complementary tools to investigate the energetics of biomolecular recognition
    Journal of Molecular Recognition, 1999
    Co-Authors: Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    The principles of Isothermal Titration Calorimetry (ITC) and differential scanning Calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters ΔG, ΔH, ΔS, and ΔCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation. Copyright © 1999 John Wiley & Sons, Ltd.

  • Isothermal Titration Calorimetry and differential scanning Calorimetry as complementary tools to investigate the energetics of biomolecular recognition
    Journal of Molecular Recognition, 1999
    Co-Authors: Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    The principles of Isothermal Titration Calorimetry (ITC) and differential scanning Calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters DeltaG, DeltaH, DeltaS, and DeltaCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation.

Peter Schuck - One of the best experts on this subject based on the ideXlab platform.

  • integration and global analysis of Isothermal Titration Calorimetry data for studying macromolecular interactions
    Nature Protocols, 2016
    Co-Authors: Chad A Brautigam, Carolyn Vargas, Sandro Keller, Huaying Zhao, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a Titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single Titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric Titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.

  • Integration and global analysis of Isothermal Titration Calorimetry data for studying macromolecular interactions
    Nature protocols, 2016
    Co-Authors: Chad A Brautigam, Carolyn Vargas, Sandro Keller, Huaying Zhao, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a Titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single Titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric Titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.

  • high precision Isothermal Titration Calorimetry with automated peak shape analysis
    Analytical Chemistry, 2012
    Co-Authors: Sandro Keller, Carolyn Vargas, Chad A Brautigam, Huaying Zhao, Grzegorz Piszczek, Peter Schuck
    Abstract:

    Isothermal Titration Calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noi...