Protein-protein interaction dynamics by amide H/2H exchange mass spectrometry

Amide H/2H exchange detected by mass spectrometry provides a powerful tool for observing changes that occur upon protein-protein interactions. In general, it is possible to observe protection of surface amides, they become less solvent exposed when they are buried at the interface. The information thus obtained about the location of the protein-protein interface is useful for building a correct docked structure of the protein-protein complex. Examples of protein-protein interfaces that were correctly identified by such methods include the thrombin-thrombomodulin interaction and the interaction between the regulatory and catalytic subunit of protein kinase A. Amide exchange also affords a view into the subtle changes in the ensemble of states that occur upon protein modification or protein-protein binding. Examples of proteins in which amide exchange has been used to observe phosphorylation-induced changes include ERK2 and CheB. Amide exchange showed the pathway of communication between the cAMP-binding site and the catalytic subunit site within the regulatory subunit of protein kinase A. Clues as to how thrombomodulin regulates the catalytic activity of thrombin were also obtained.