Analysis of cooperativity by isothermal titration calorimetry

Abstract

Cooperative binding pervades Nature. This review discusses the use of isothermal titration calorimetry (ITC) in the identification and characterisation of cooperativity in biological interactions. ITC has broad scope in the analysis of cooperativity as it determines binding stiochiometries, affinities and thermodynamic parameters, including enthalpy and entropy in a single experiment. Examples from the literature are used to demonstrate the applicability of ITC in the characterisation of cooperative systems.

Keywords: NMR; cooperativity; global analysis; isothermal titration calorimetry; multiprotein complexes; stoichiometry; thermodynamics.

Figure 1.

Reaction scheme for the binding of heterogeneous ligands, X and Y, to a macromolecule, M, containing two binding sites. The stepwise association constants, K_X and K_Y, for the binding of ligands X and Y respectively, to free macromolecule are shown, as the association constants for the binding of ligands to preformed complexes, K_X|Y and K_Y|X.

Figure 2.

Global and cooperative thermodynamic parameters associated with the negatively cooperative binding of Fd to FNR-NADP⁺.

Figure 3.

The three distinguishable types of ligand binding sites: isolated (iso), singly contiguous (sc) and doubly contiguous (dc). The potential number of binding sites is given by (N − l + 1), so that in this example where N = 6 and l = 2, there are five potential binding sites.

Figure 4.

The crystal structure of chromatin protein Sac7d bound to a nucleic acid fragment (PDB ID: 1AZP) demonstrates that binding induces a 66° kink in the structure of DNA. Thermodynamics of the binding event determined by application of the non-cooperative McGhee–von Hippel model to ITC data demonstrates that binding is characterised by a favourable entropy term and an unfavourable enthalpy term attributed to the energetic penalty of kinking DNA.