Authors: Giorgio Pochetti & Roberta Montanari
It could be very useful in drug development to determine the relative affinities of a ligand that can bind to a protein with two (or more) different and specific binding sites.
X-ray crystallography is a very powerful technique to determine the locations and describe the features of each binding site but it is unuseful to establish their relative affinities. Moreover, the stoichiometry of binding observed in the crystal structure of a complex could be an artifact of the crystallization conditions.
On the contrary, isothermal titration calorimetry (ITC) can be used to assign the correct stoichiometry of the association, depending on the initial and final concentration ratio between ligand and protein, and to establish the affinity for each specific binding site, if combined with structural information from X-ray crystallography.