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.
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.
Abe
Masato
M
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.
Ohno
Satoshi
S
Yokogawa
Takashi
T
Nakanishi
Takeshi
T
Arisaka
Fumio
F
Hosoya
Takamitsu
T
Hiramatsu
Toshiyuki
T
Suzuki
Masaaki
M
Ogasawara
Tomio
T
Sawasaki
Tatsuya
T
Nishikawa
Kazuya
K
Kitamura
Masaya
M
Hori
Hiroyuki
H
Endo
Yaeta
Y
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
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