Photocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA Complexes

Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.