title: Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing.
abstract: A cell-free protein synthesis system is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. Here, the authors describe unnatural amino acid probing in a wheat germ cell-free translation system as a method for detecting the structural changes that occur in a cofactor binding protein on a conversion of the protein from an apo-form to a holo-form. The authors selected the FMN-binding protein from Desulfovibrio vulgaris as a model protein. The apo-form of the protein was synthesized efficiently in the absence of FMN. The purified apo-form could be correctly converted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chromatography, analytical ultracentrifugation, and circular dichroism-spectra studies suggested that the FMN-binding site of the apo-form is open as compared with the holo-form. To confirm this idea, the unnatural amino acid probing was performed by incorporating 3-azido-L-tyrosine at the Tyr35 residue in the FMN-binding site. The authors optimized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triarylphosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 residue in the apo-form is open to solvent.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Abe
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohno
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokogawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Fumio
authors.initials: F
authors.lastname: Arisaka
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: Tomio
authors.initials: T
authors.lastname: Ogasawara
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Masaya
authors.initials: M
authors.lastname: Kitamura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002474:Cell-Free System; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D008954:Models, Biological; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000378:metabolism; D014908:Triticum / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001386:Azides; D001426:Bacterial Proteins; C086046:FMN-binding protein, Desulfovibrio vulgaris; D005420:Flavoproteins; D012365:RNA, Transfer, Tyr; C065394:3-azidotyrosine; D014443:Tyrosine; D019793:Fluorescein