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 Masato Abe, 1 Satoshi Ohno, 2 Takashi Yokogawa, 2 Takeshi Nakanishi, 3 Fumio Arisaka, 4 Takamitsu Hosoya, 5 Toshiyuki Hiramatsu, 5 Masaaki Suzuki, 5 Tomio Ogasawara, 6 Tatsuya Sawasaki, 1,6,7 Kazuya Nishikawa, 2 Masaya Kitamura, 3 Hiroyuki Hori, 1,6 * and Yaeta Endo 1,6,7 1 Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan 2 Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu 501-1193, Japan 3 Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan 4 Department of Molecular and Cellular Assembly, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan 5 Division of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu 501-1194, Japan 6 Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan 7 Cell-free Science Technology Research Center, Ehime University, Matsuyama 790-8577, Japan ABSTRACT A cell-free protein synthesis sys- tem 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 bind- ing 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 con- verted to the holo-form. Thus, the system could synthesize the active apo-form. Gel filtration chro- matography, 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 opti- mized three steps in their system. The introduced 3-azido-L-tyrosine residue was subjected to specific chemical modification by a fluorescein-triaryl- phosphine derivative. The initial velocity of the apo-form reaction was 20 fold faster than that of the holo-form, demonstrating that the Tyr35 resi- due in the apo-form is open to solvent. Proteins 2007;67:643–652. V V C 2007 Wiley-Liss, Inc. Key words: cell-free translation; unnatural amino acid; chemical modification; flavin; fla- voprotein; FMN-binding protein INTRODUCTION A cell-free protein synthesis system 1–7 is a powerful tool with which unnatural amino acids can be introduced into polypeptide chains. 8–11 Indeed, several cell-free sys- tems have been utilized to incorporate selenomethionine for X-ray crystal structure analyses 6,12,13 and stable iso- tope labeling for NMR studies. 6,14,15 However, such structural studies generally require professional techni- ques and expensive instruments, are considerably time- consuming, and incur high costs. Nowadays, numerous proteins have been structurally studied and their coordi- nates are available from the Protein Data Bank. 16 In a general laboratory, quick, convenient, and inexpensive methods based on these structural data are desirable to detect structural change(s), protein–protein interactions, and/or protein–small molecule(s) interactions. Our cell- free system is derived from wheat germ extract from which translation inhibitors, such as ribosome inactiva- Abbreviations: FMN-BP, FMN-binding protein; azTyr, 3-azido-L- tyrosine. Grant sponsor: Grant-in-aid for Science Research on Priority Areas; Grant number: 18048032; Grant sponsor: Grant-in-aid for Science Research from the Ministry of Education, Science, Sports, and Culture of Japan; Grant number: 17613003; Grant sponsor: Japan Society for the Promotion of Science; Grant number: JSPS- RFTF 96100305. *Correspondence to: Hiroyuki Hori, Department of Applied Chem- istry, Faculty of Engineering, Ehime University, Bunkyo 3, Mat- suyama 790-8577, Japan. E-mail: hori@eng.ehime-u.ac.jp Received 8 September 2006; Revised 14 November 2006; Accepted 20 November 2006 Published online 8 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21341 V V C 2007 WILEY-LISS, INC. PROTEINS: Structure, Function, and Bioinformatics 67:643–652 (2007)