MASS-SPECTROMETRIC CHARACTERIZATION OF A PROTEIN LIGAND INTERACTION

Src homology 2 (SH-2) domains are found in a variety of protein kinase s and are believed to be recognition sites for specific phosphotyrosin e-containing peptide sequences. We have investigated the deuterium exc hange behavior of a recombinant SH-2 domain, using electrospray ioniza tion mass spectrometry to monitor the incorporation of deuterium. In t he presence of a tight-binding phosphopeptide, the exchange slows dram atically. Because the NMR and X-ray crystal structures of the protein do not indicate a large conformational shift upon binding, we interpre t the ESI-MS results as an increase in conformational stability. Phosp hoserine or phosphothreonine sequences do not show the effect; neither does an un-phosphorylated analogue or a shorter peptide containing ph osphotyrosine. The protein can be titrated with ligand by monitoring t he exchange behavior to give the stoichiometry of the complex. Ions fr om a noncovalent complex of SH-2 and the phosphopentapeptide exhibit t he same exchange kinetics as those of the uncomplexed protein in the p resence of ligand.