Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine
Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine
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DOI：
10.1093/jb/mvm036
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发表时间：
2007-03-01
影响因子：
2.7
通讯作者：
Nishikawa, Kazuya
中科院分区：
文献类型：
--
作者：
Ohno, Satoshi;Matsui, Megumi;Nishikawa, Kazuya
利用非天然氨基酸3-叠氮基酪氨酸对蛋白质进行位点选择性修饰的有效方法已经发展起来。该方法利用酵母琥珀抑制基因tRNA（Tyr）/突变的酪氨酰-tRNA合成酶对作为大肠杆菌无细胞翻译系统中3-叠氮基酪氨酸的载体，并利用三芳基膦衍生物对叠氮基进行特异性修饰。使用大鼠钙调素（CaM）作为模型蛋白，我们制备了几个非天然的CaM分子，每个携带叠氮酪氨酸在预定的位置72，78，80或100，分别。这些蛋白质的翻译后修饰与共轭化合物的三芳基膦和生物素产生的位点选择性生物素化的钙调素分子。这些蛋白质的反应效率是相似的，无论引入的位置，和位点特异性的生物素化，使用质谱法进行了确认。此外，CBP结合活性的生物素化的钙调素被证实是类似的野生型钙调素。该方法本质上是通用的，因为它应该容易地适用于引入任何其它所需的化合物（例如，探针和交联剂）插入蛋白质的选定位点，只要可以化学合成三芳基膦的适当衍生化合物。通过利用这些位点选择性修饰的蛋白质，将极大地促进蛋白质功能和蛋白质-蛋白质网络的分子机制的阐明。
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.