title: Measuring the Kinetics of Molecular Association by Isothermal Titration Calorimetry.
abstract: The real-time power response inherent in an isothermal titration calorimetry (ITC) experiment provides an opportunity to directly analyze association kinetics, which, together with the conventional measurement of thermodynamic quantities, can provide an incredibly rich description of molecular binding in a single experiment. Here, we detail our application of this method, in which interactions occurring with relaxation times ranging from slightly below the instrument response time constant (12.5 s in this case) to as large as 600 s can be fully detailed in terms of both the thermodynamics and kinetics. In a binding titration scenario, in the most general case an injection can reveal an association rate constant (kon). Under more restrictive conditions, the instrument time constant-corrected power decay following each injection is simply an exponential decay described by a composite rate constant (kobs), from which both kon and the dissociation rate constant (koff) can be extracted. The data also support the viability of this exponential approach, for kon only, for a slightly larger set of conditions. Using a bimolecular RNA folding model and a protein-ligand interaction, we demonstrate and have internally validated this approach to experiment design, data processing, and error analysis. An updated guide to thermodynamic and kinetic regimes accessible by ITC is provided.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA. Electronic address: kirk.vandermeulen@wisc.edu.
authors.forename: Kirk A
authors.initials: KA
authors.lastname: Vander Meulen
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Horowitz
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Trievel
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA. Electronic address: sebutcher@wisc.edu.
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Butcher
mesh_terms: D002151:Calorimetry / Q000379:methods*; D007700:Kinetics; D011485:Protein Binding; D013816:Thermodynamics
keywords: Isothermal titration calorimetry; Kinetics; Lysine methyltransferase; RNA; SET7/9; Tetraloop receptor