MASS-SPECTROMETRIC CHARACTERIZATION OF A PROTEIN LIGAND INTERACTION

Src homology 2 (SH-2) domains are found in a variety of protein kinases and are believed to be recognition sites for specific phosphotyrosine-containing peptide sequences. We have investigated the deuterium exchange behavior of a recombinant SH-2 domain, using electrospray ionization mass spectrometry to monitor the incorporation of deuterium. In the presence of a tight-binding phosphopeptide, the exchange slows dramatically. Because the NMR and X-ray crystal structures of the protein do not indicate a large conformational shift upon binding, we interpret the ESI-MS results as an increase in conformational stability. Phosphoserine or phosphothreonine sequences do not show the effect; neither does an un-phosphorylated analogue or a shorter peptide containing phosphotyrosine. The protein can be titrated with ligand by monitoring the exchange behavior to give the stoichiometry of the complex. Ions from a noncovalent complex of SH-2 and the phosphopentapeptide exhibit the same exchange kinetics as those of the uncomplexed protein in the presence of ligand.