4 years ago

Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes in vitro and in vivo

Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes in vitro and in vivo
Daniel Horn-Ghetko, Matthias P. Müller, Roger S. Goody, Marie-Kristin von Wrisberg, Aymelt Itzen, Maximilian Fottner, Kathrin Lang, Marko Cigler, Thorsten G. Müller
The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein–protein interactions in vitro and in vivo. Triggered by proximity: Site-specific incorporation of structurally flexible unnatural amino acids allows the covalent trapping of low-affinity and transient protein–protein interactions under native conditions. This crosslinking approach can be used to aid the co-crystallization and structure elucidation of low-affinity protein complexes.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201706927

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