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Analyses of Protein–Protein Interactions by In Vivo Photocrosslinking in Budding Yeast

  • Takuya Shiota
  • Shuh-ichi Nishikawa
  • Toshiya Endo
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1033)

Abstract

Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-l-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein–protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed for pBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein–protein interactions of the target proteins at work.

Key words

Protein interactions Photocrosslink Unnatural amino acids Suppressor tRNA Yeast 

Notes

Acknowledgments

We thank Dr. Peter G. Schultz for providing the p-6xtRNA plasmids. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and a grant from the Japan Science and Technology Corporation (JST).

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Takuya Shiota
    • 1
  • Shuh-ichi Nishikawa
    • 1
  • Toshiya Endo
    • 1
  1. 1.Department of ChemistryNagoya UniversityNagoyaJapan

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