Dissecting complex binding equilibria involving protein–protein and protein–metal interactions

Isothermal titration calorimetry (ITC) is widely used in drug discovery and in structural biology to characterize specific interactions occurring between molecules in aqueous solutions. The possibility to run the experiments in solution conditions, without modifying the system under analysis, and to obtain all thermodynamic parameters in a single experiment makes this technique a “gold standard” for the characterization of binding reactions involving biological molecules. Biological reactions are often associated with side reactions, such as conformational changes and oligomerization, and sometimes include multiple reacting species present in solution. This may produce complicated binding isotherm that needs to be interpreted as complex binding schemes. In this case, applying the Occam's razor principle is recommended, using the simplest model able to fit the data and coherent with information deriving from other experimental techniques. When this procedure is done properly, ITC can produce unique information for the dissection and interpretation of molecular mechanisms and perfectly lies in the pipeline of structural biology projects. In this chapter, three cases in which ITC, used as a complementary tool to other biochemical and biophysical techniques, was essential to dissect multiple concomitant equilibria that generated multipartite isotherms are described. These examples evidence the synergic potential of ITC and other experimental approaches and show how to get a full comprehension of the system of interest at the molecular level requires working at different levels, complementing diverse technological tools.