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Journal of Biochemistry Advance Access originally published online on January 3, 2007
Journal of Biochemistry 2007 141(3):335-343; doi:10.1093/jb/mvm036
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© 2007 The Japanese Biochemical Society.

Site-selective Post-translational Modification of Proteins Using an Unnatural Amino Acid, 3-Azidotyrosine

Satoshi Ohno1,*, Megumi Matsui1, Takashi Yokogawa1, Masashi Nakamura1, Takamitsu Hosoya2,{dagger}, Toshiyuki Hiramatsu2,{dagger}, Masaaki Suzuki2, Nobuhiro Hayashi3 and Kazuya Nishikawa1

1Department of Biomolecular Science, Faculty of Engineering; 2Division of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1193; and 3Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan

*To whom correspondence should be addressed. Tel: +81-58-293-2645, Fax: +81-58-230-1893, E-mail: ohno{at}gifu-u.ac.jp

Received October 5, 2006; Accepted December 20, 2006


  Abstract

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNATyr/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

Key Words: azidotyrosine, modification, protein synthesis, suppression, tyrosyl-tRNA synthetase

Abbreviations: aaRS, aminoacyl-tRNA synthetase; CaM, calmodulin; CBP-ECFPht, ECFP having N-terminal CaM-binding–peptide and C-terminal hexa-histidine; ECFP, enhanced cyan fluorescent protein, one of the enhanced green fluorescent protein colour variants; TyrRS, tyrosyl-tRNA synthetase; EC. 6. 1. 1. 1.; Y43G, yeast TyrRS with a replacement of tyrosine 43 by glycine

{dagger} Present address: Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.


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