Current applications of isothermal titration calorimetry to the study of protein complexes (original) (raw)
Related papers
Methods in Cell Biology, 2008
Abstract Isothermal titration calorimetry (ITC) is now routinely used to directly characterize the thermodynamics of biopolymer binding interactions and the kinetics of enzyme-catalyzed reactions. This is the result of improvements in ITC instrumentation and data analysis software. Modern ITC instruments make it possible to measure heat eVects as small as 0.1 mcal (0.4 mJ), allowing the determination of binding constants, K's, as large as 10 8 -10 9 M À1 . Modern ITC instruments make it possible to measure heat rates as small as 0.1 mcal/sec, allowing for the precise determination of reaction rates in the range of 10 À12 mol/sec. Values for K m and k cat , in the ranges of 10 À2 -10 3 mM and 0.05-500 sec À1 , respectively, can be determined by ITC. This chapter reviews the planning of an optimal ITC experiment for either a binding or kinetic study, guides the reader through simulated sample experiments, and reviews analysis of the data and the interpretation of the results.
Analytical biochemistry, 2015
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 give...
Biological Applications of Isothermal Titration Calorimetry
Physical Chemistry Research, 2015
Most of the biological phenomena are influenced by intermolecular recognition and interaction. Thus, understanding the thermodynamics of biomacromolecule ligand interaction is a very interesting area in biochemistry and biotechnology. One of the most powerful techniques to obtain precise information about the energetics of (bio) molecules binding to other biological macromolecules is isothermal titration calorimetry (ITC). In a typical ITC experiment, a macromolecule solution is titrated by a solution containing a reactant at a constant temperature, and exchanged heat of the reaction is measured, allowing determination of thermodynamic parameters (enthalpy change, entropy change, change in Gibbs free energy, binding affinity and stoichiometry) of molecular interactions. In this review article, we describe the ITC approach briefly and review some applications of ITC for studying protein-ligand interactions, protein-protein interactions, self-association, and drug design processes. Fu...
Multithermal titration calorimetry: A rapid method to determine binding heat capacities
Biophysical Chemistry, 2006
Herein a new method that allows binding DCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature readjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme -chitotriose complex as a model system. D
Isothermal titration calorimetry of membrane proteins — Progress and challenges
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014
Integral membrane proteins, including G protein-coupled receptors (GPCR) and ion channels, mediate diverse biological functions that are crucial to all aspects of life. The knowledge of the molecular mechanisms, and in particular, the thermodynamic basis of the binding interactions of the extracellular ligands and intracellular effector proteins is essential to understand the workings of these remarkable nanomachines. In this review, we describe how isothermal titration calorimetry (ITC) can be effectively used to gain valuable insights into the thermodynamic signatures (enthalpy, entropy, affinity, and stoichiometry), which would be most useful for drug discovery studies, considering that more than 30% of the current drugs target membrane proteins. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
Isothermal titration calorimetry in membrane protein research
Journal of Pharmaceutical and Biomedical Analysis, 2014
Isothermal titration calorimetry is a versatile method to characterize energetics of intermolecular reactions and in particular interactions between drug molecules and their macromolecular targets. The assay is widely used in medicinal chemistry to quantification and characterization of molecular mechanisms of these interactions. The article reviews applications of the method to study ligand interactions with various classes of embedded membrane proteins. These proteins constitute very important fraction of macromolecular targets for medicinal compounds used in current therapies and recently emerged for detailed structural, functional and biochemical characterization of their interactions with drug molecules.