Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker

In this paper, we describe the efficient synthesis of a diazirine cross-linker (photo-Met) and demonstrate that this amino acid is compatible with solid-phase peptide synthesis and expressed protein ligation (EPL). Accordingly, we have exploited the unique power of semi-synthesis to incorporate multiple different probes into the same protein by preparing a version of Smad2 signaling protein containing both photo-Met and an activating phospho-serine. Through judicious placement of the cross-linker, we have been able to covalently capture a transient MH2-MH2 interaction that is dependent upon the PTM in the protein.