title: Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine.
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 tRNA(Tyr)/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.
authors.affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. ohno@gifu-u.ac.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohno
authors.forename: Megumi
authors.initials: M
authors.lastname: Matsui
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokogawa
authors.forename: Masashi
authors.initials: M
authors.lastname: Nakamura
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hosoya
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Hiramatsu
authors.forename: Masaaki
authors.initials: M
authors.lastname: Suzuki
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Hayashi
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishikawa
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry*; D019921:Biotinylation / Q000379:methods; D002147:Calmodulin / Q000737:chemistry*; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
chemical_list: D001386:Azides; D002147:Calmodulin; D009943:Organophosphorus Compounds; D012365:RNA, Transfer, Tyr; C065394:3-azidotyrosine; C061896:triphenylphosphine; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase