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Journal of Biochemistry Advance Access published online on January 3, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm036
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© 2006 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, and 2Division of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan; 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-azido-tyrosine has been developed. This method utilizes the yeast amber suppressor tRNATyr/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azido-tyrosine in an E. coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido-group. Using rat calmodulin as a model protein, we prepared several unnatural calmodulin molecules, each carrying an azido-tyrosine at predetermined positions 72, 77, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated calmodulin 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 calmodulins was confirmed to be similar to that of wild type calmodulin. 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: Azido-tyrosine, Modification, Protein Synthesis, suppression, Tyrosyl-tRNA synthetase

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


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