All functions of living organisms are regulated by sophisticated signaling networks that involve a multitude of interactions between different proteins. Understanding the topology of protein-protein complexes provides important clues for the development of molecules able to interfere with protein interactions, allowing us to modulate signaling pathways for therapeutic purposes. Protein complexes extracted from the cell and reconstituted in appropriate artificial environments can be characterized at high resolution by NMR and crystallography techniques. Alternative methods are needed both to gain information from the natural context of the live cell and to investigate protein targets that elude direct structural characterization.
We focus on mapping protein-protein interaction surfaces in the live cell by combining chemical tools and modern molecular biology techniques. In particular, we use the expanded genetic code technology to incorporate crosslinking amino acids into proteins at specific sites. The unnatural moiety is built into the protein bait directly in the live cell through regular ribosomal synthesis, without the need of any additional step in vitro. The crosslinker captures interacting protein partners that come within its range of proximity. Systematic incorporation of the probe throughout interaction domains provides panoramic information about the topology of the interaction surfaces.