Isothermal titration calorimetry to determine the association constants for a ligand bound simultaneously to two specific protein binding sites with different affinities

Authors

Giorgio Pochetti and Roberta Montanari

Summary

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. In this protocol a direct and reverse titration is performed to determine the stoichiometry of the ligand binding. A third titration is necessary, after pre-equilibration of the protein with another ligand that is known (from X-ray crystallography) to bind univocally to only one out of the two sites, to assign the association constants for each site.

Further details

The protocol was published on Protocol Exchange on 20 December 2012. To see the entire protocol, click on the source link.

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