Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Abstract

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given. Video LinkThe video component of this article can be found at https://www.jove.com/video/2796/ ProtocolIsothermal titration calorimetry (ITC) is a well established technique that can determine all the thermodynamic parameters (affinity, enthalpy and stoichiometry) of a binding interaction in one experiment.1 ITC works by titrating one reactant into a second reactant under isothermal conditions. The signal measured is the heat released or absorbed upon interaction (binding) of the two reactants. A series of injections are performed and the heat signal will approach zero as the limiting reactant becomes saturated. Fitting of the isotherm gives the thermodynamic parameters. Several reviews are available that describe the instrumentation as well as the math of data collection and analysis. 2,3 While other calorimeters are available (most notably the ITC 200 with small volumes), here we describe a general protocol for the VP-ITC manufactured by MicroCal (now part of GE Healthcare).

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

“…kinetics). In particular, isothermal titration calorimetry (ITC) has been adopted as method of choice to characterize the thermodynamics of biomolecular equilibria, involving protein-ligand, protein-protein, protein-metal ions and protein-DNA interactions [1][2][3][4][5][6] . In addition, the ability of ITC to provide kinetic information makes it a very powerful system to measure enzyme catalysis, although the potential of this application is still underestimated [7][8][9] .…”

High Force Catch Bond Mechanism of Bacterial Adhesion in the Human Gut

“…The titration was carried out at 25 °C using a VP-ITC instrument 73 . The analyte was 16.1 µM Coh (lacking ELP linker and ddFLN4 domains) and the injectant was 126 µM XMod-Doc protein (lacking ELP linker and ddFLN4 domains).…”

Differential Radial Capillary Action of Ligand Assay (DRaCALA)

“…Serial addition of a known concentration of ligand to a known concentration of protein allows for calculation of binding affinity, stoichiometry of binding, and enthalpy of binding. While powerful, ITC is sensitive to subtle differences in the buffers used for proteins and nucleic acids (Duff, Grubbs, & Howell, 2011;Wiseman, Williston, Brandts, & Lin, 1989). Furthermore, ITC typically requires a relatively large amount of protein and ligand for assaying their interactions.…”