Exploring the Binding Interaction Between Phosphotyrosine Peptides and SH2 Domains by Proximal Crosslinking

Proximal crosslinking refers to the site-specific conjugation reaction between a synthetic ligand with a bioorthogonal reactive group incorporated at a particular site and a protein of interest (POI). The binding interaction positions a reactive group of a native amino acid of the POI to the proximity of the reactive group in the ligand. The covalent conjugation increases the molecular weight of the POI, shows an upshift in the polyacrylamide gel, and gives a fluorescent band if the ligand is fluorescently labeled. Here, we summarize a method to covalently conjugate phosphotyrosine peptides and SH2 domains that contain cysteine residues. This method yields covalent peptide blockers for a set of SH2 proteins and elucidates the binding interaction between phosphotyrosine peptides and SH2 domains.