Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 56, Issue 49, Pages 15737-15741Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201706927
Keywords
covalent stabilization; protein-protein interactions; protein crosslinking; proximity effects; unnatural amino acids
Categories
Funding
- Excellence Initiative
- EU Marie Curie COFUND Program
- Fonds der Chemischen Industrie
- [SFB1035]
- [GRK1721]
- [SPP1623]
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The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes invivo 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 invivo 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 invitro and invivo.
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