Covalently Capturing Protein Interactions in Living Cells by Site-Specific Incorporation of Photo-Cross-Linkable Amino Acids

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Abstract

Protein functions in living cells are strictly regulated to precisely execute cellular processes, which are accomplished by the interaction of proteins in a weak and transient manner. Protein photo-cross-linking is a powerful way to stabilize such interactions by covalently bonding the interacting proteins to each other. However, the in vivo use of low molecular weight cross-linkers has been very limited owing to the difficulty of creating specific modifications of the desired proteins for many cellular proteins. Recent developments in genetic code expansion technology have overcome such difficulties to create a variety of photoreactive moieties that can be site-specifically incorporated into the desired protein as unnatural amino acids in living cells. In this chapter, we present an overview of in vivo protein photo-cross-linking with genetically encoded photo-cross-linkable amino acids (PAAs) and then discuss its application for covalently capturing extremely weak and transient interactions, identification of direct binding partners of a target protein, and precise mapping of the binding interface between interacting proteins under physiologically relevant conditions.

Keywords

Protein–protein interaction Protein complex Cross-link Unnatural amino acid Genetic code expansion 
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Copyright information

© Springer Japan KK 2017

Authors and Affiliations

  1. 1.Laboratory of Molecular Medicine, Graduate School of Pharmaceutical SciencesOsaka UniversitySuitaJapan
  2. 2.Division of Structural and Synthetic BiologyRIKEN Center for Life Science TechnologiesYokohamaJapan

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