Isothermal Titration Calorimetry

Velázquez‐Campoy, Adrián; Ohtaka, Hiroyasu; Nezami, Azin; Muzammil, Salman; Freire, Ernesto

doi:10.1002/0471143030.cb1708s23

Cited by 180 publications

(164 citation statements)

References 47 publications

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“…Here we will concentrate on the application of (ITC) and the (TSA). Both methods have been described previously in detail, especially ITC [Freyer and Lewis, 2008, Landbury, 2004, Velazquez-Campoy et al, 2004. However, TSA is rather unconventional and underused despite its great advantages and usefulness , Cimmperman et al, 2008, Matulis et al, 2005.…”

Section: Thermodynamics Of Binding By Isothermal Titration Calorimetrmentioning

confidence: 99%

Thermodynamics of Natural and Synthetic Inhibitor Binding to Human Hsp90

Petrikaitė¹,

Matulis²

2011

Application of Thermodynamics to Biological and Materials Science

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Section: Thermodynamics Of Binding By Isothermal Titration Calorimetrmentioning

confidence: 99%

Thermodynamics of Natural and Synthetic Inhibitor Binding to Human Hsp90

Petrikaitė¹,

Matulis²

2011

Application of Thermodynamics to Biological and Materials Science

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“…Isothermal Titration Calorimetry (ITC) is a wellestablished technique for analysis of protein-ligand interactions to measure accurate binding affinity constants and establishing the reaction stoichiometry [1][2][3][4] . The ITC is frequently used to complement Nuclear Magnetic Resonance (NMR) studies to provide thermodynamic information on complex interaction mechanisms [5][6][7] .…”

Section: Introductionmentioning

confidence: 99%

“…Simulations demonstrate that the mechanisms where the free or ligand-bound protein undergoes dimerization (such that the ligand cannot bind to or dissociate from the dimer) produce very distinctive titration profiles. In contrast, profiles of the pre-existing equilibrium or induced-fit models cannot be distinguished from a simple two-state process, requiring data from additional techniques to positively identify these mechanisms.Isothermal Titration Calorimetry (ITC) is a wellestablished technique for analysis of protein-ligand interactions to measure accurate binding affinity constants and establishing the reaction stoichiometry [1][2][3][4] . The ITC is frequently used to complement Nuclear Magnetic Resonance (NMR) studies to provide thermodynamic information on complex interaction mechanisms [5][6][7] .…”

mentioning

confidence: 99%

Resolving Three-State Ligand-Binding Mechanisms by Isothermal Titration Calorimetry: A Simulation Study

Kovrigin

2017

Preprint

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ABSTRACT:In this paper, I theoretically analyzed ITC profiles for three-state equilibria involving ligand binding coupled to isomerization or dimerization transitions. Simulations demonstrate that the mechanisms where the free or ligand-bound protein undergoes dimerization (such that the ligand cannot bind to or dissociate from the dimer) produce very distinctive titration profiles. In contrast, profiles of the pre-existing equilibrium or induced-fit models cannot be distinguished from a simple two-state process, requiring data from additional techniques to positively identify these mechanisms.Isothermal Titration Calorimetry (ITC) is a wellestablished technique for analysis of protein-ligand interactions to measure accurate binding affinity constants and establishing the reaction stoichiometry [1][2][3][4] . The ITC is frequently used to complement Nuclear Magnetic Resonance (NMR) studies to provide thermodynamic information on complex interaction mechanisms [5][6][7] . I previously reported analysis of NMR line shapes in titrations of systems with protein-ligand interactions coupled to isomerization or dimerization equlibria 8 . In this communication I am outlining major patterns one can expect in such three-state coupled equilibria from ITC experiments to help NMR and ITC experimentalists anticipate results from real systems. MethodsAll simulations were performed using LineShapeKin Simulation software, which is designed to simulate NMR line shapes and ITC titration profiles. LineShapeKin Simulation was described in my previous report 8 and is freely available from http://lineshapekin.net. The models under discussion in this paper are given in Fig. 1, where R stands for a "receptor", which is a protein or nucleic acid or any other large or small molecule. The "L" stands for a ligand, which may as well be any of the above-mentioned molecules. We only need to discriminate R and L because in ITC one of the binding partners resides in the calorimeter cell, while another is added from the syringe. I am assigning "R" to the species in the cell and will casually refer to it as a "protein" while its binding partner, L, will be in a syringe and will be called a "ligand". The reader must appreciate that this assignment is arbitrary and completely reversible. In fact, if one of the binding partners is poorly soluble it is advisable to place it in the calorimeter cell because the solution in the syringe has to be 10-20 times more concentrated. Such "reverse titrations" are described by the U-L and U-L2 models, which are equivalent to U-R and U-R2, therefore, will be dropped for clarity. Specific parameters for calculations of ITC profiles discussed in this paper are given in the Appendix. Fig. 1 The three-state models evaluated in this study. Model names (according to a convention of the LineShapeKin Simulation) are given in red. Binding and coupled transitions labels are blue. Results and DiscussionFig. 1 summarizes the models that one can use to describe a protein molecule binding a single ligand and undergoing a one-st...

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“…If the binding reaction is stoichiometric and inhibits the enzyme, then both approaches should yield similar results. In this study, the enzymatic inhibition method was compared with the results obtained using two biophysical techniques, isothermal titration calorimetry [3] and a thermal shift assay (ThermoFluor, differential scanning fluorimetry) [4]. The inhibition methods are more widely used than the biophysical methods, but determining the binding reactions by several techniques will reduce the uncertainty in the measurements.…”

Section: Introductionmentioning

confidence: 99%