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.
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References
Ellman J, Mendel D, Anthony-Cahill S et al (1991) Biosynthetic method for introducing unnatural amino acids site-specifically into proteins. Methods Enzymol 202:301–336
Dormán G, Prestwich GD (1994) Benzophenone photophores in biochemistry. Biochemistry 33:5661–5673
Farrell IS, Toroney R, Hazen JL et al (2005) Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Nat Methods 2:377–384
Chin JW, Cropp TA, Anderson JC et al (2003) An expanded eukaryotic genetic code. Science 301:964–967
Hino N, Okazaki Y, Kobayashi T et al (2005) Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nat Methods 2:201–216
Shiota T, Mabuchi H, Tanaka-Yamano S et al (2011) In vivo protein-interaction mapping of a mitochondrial translocator protein Tom22 at work. Proc Natl Acad Sci USA 108:15179–15183
Adams A, Gottschling DE, Kaiser CA et al (1998) Methods in yeast genetics, a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Shiota T, Nishikawa S, Endo T (2012) Protein interaction mapping using the in vivo site-specific photo crosslinking technique. Exp Med 30:1799–1805 (in Japanese)
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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Shiota, T., Nishikawa, Si., Endo, T. (2013). Analyses of Protein–Protein Interactions by In Vivo Photocrosslinking in Budding Yeast. In: Rapaport, D., Herrmann, J. (eds) Membrane Biogenesis. Methods in Molecular Biology, vol 1033. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-487-6_14
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DOI: https://doi.org/10.1007/978-1-62703-487-6_14
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-486-9
Online ISBN: 978-1-62703-487-6
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