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
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
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DOI：
10.1002/prot.21341
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发表时间：
2007-05-15
影响因子：
2.9
通讯作者：
Endo, Yaeta
中科院分区：
文献类型：
--
作者：
Abe, Masato;Ohno, Satoshi;Endo, Yaeta
无细胞蛋白质合成系统是一种强大的工具，可以将非天然氨基酸引入多肽链中。在这里，作者描述了小麦胚芽无细胞翻译系统中的非天然氨基酸探测，作为检测辅因子结合蛋白在蛋白质从脱辅基形式转化为全息形式时发生的结构变化的方法。作者选择来自普通脱硫弧菌的 FMN 结合蛋白作为模型蛋白。在没有 FMN 的情况下，可以有效合成该蛋白的 apo 形式。纯化的apo-形式可以正确地转化为holo-形式。因此，该系统可以合成活性apo-形式。凝胶过滤色谱、分析超速离心和圆二色光谱研究表明，与全息形式相比，apo 形式的 FMN 结合位点是开放的。为了证实这一想法，通过在 FMN 结合位点的 Tyr35 残基处掺入 3-叠氮基-L-酪氨酸来进行非天然氨基酸探测。作者优化了系统中的三个步骤。引入的3-叠氮基-L-酪氨酸残基通过荧光素-三芳基膦衍生物进行特定的化学修饰。 apo 型反应的初始速度比全型反应的初始速度快 20 倍，这表明 apo 型中的 Tyr35 残基对溶剂是开放的。
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.