Chemical crosslinking is well-established for investigating protein-protein interactions. Photocrosslinking, a classical technique, is broadly used, however, selectivity and UV-toxicity problems appear in a biological context. In search for a novel methodology for efficient crosslinking, our research group recently developed the furan crosslinking technology for oligonucelotides1-6, further applicable to the investigation of peptide-protein interactions7,8. Furan, a small aromatic compound, is incorporated in peptides as a 2-furyl-L-alanine and can be oxidized to a reactive aldehyde by cytochrome P450 catalysis in a natural setting1, subsequent reaction with sulfhydryl and amine groups in the protein binding partner can occur if sufficiently proximal. Until now, furan moiety was incorporated in DNA and RNA strands for subsequent duplex interstrand crosslink formation upon activation of the furan moiety by oxidation using N-bromo succinimide or singlet oxygen, and for crosslinking of RNA to proteins. Here, we have developed and optimized a crosslinking technique for Biotinylated-Furan-modified peptides to interact with GPCR proteins on live cells with spontaneous endogenous oxidation of the furan moiety moiety through an endogenous cellular mechanism.
The ligand-receptor covalent crosslinking methodology is illustrated on the Kisspeptin-10/GPR54 system. Kisspeptin is a metastasis suppressor and influences cancer cell invasion9. A Biotinylated-Furan-modified kisspeptin-10 ligand covalently couples to its glycosylated membrane receptor upon mere incubation with live cells expressing the receptor at endogenous levels. Moreover, we propose that reactive oxygen species produced by NADPH oxidases enzymes form the cellular source establishing furan oxidation7.