Generating Permissive Site-Specific Unnatural Aminoacyl-tRNA Synthetases
Generating Permissive Site-Specific Unnatural Aminoacyl-tRNA Synthetases
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DOI：
10.1021/bi901947r
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发表时间：
2010-03-02
期刊：
影响因子：
2.9
通讯作者：
Mehl, Ryan A.
中科院分区：
文献类型：
--
作者：
Miyake-Stoner, Shigeki J.;Refakis, Christian A.;Mehl, Ryan A.
基因整合非天然氨基酸（UAA）技术是一种强大的工具，可以大大提高我们研究和设计生物系统的能力。使用这些技术，研究人员可以通过引入UAA的新R基团来精确控制蛋白质中新化学部分的位置和数量，这些基团在蛋白质的一级结构中遗传编码。天然氨酰-tRNA合成酶（阿尔斯）的底物识别特性必须被修饰以将UAA掺入蛋白质中。这样做的协议在技术上很简单，但需要时间和优化，这大大限制了这一重要技术的可访问性。目前，工程化的非天然氨酰-tRNA合成酶（UaaRS）根据其翻译效率（其允许UAA掺入蛋白质的程度）和保真度（其阻止天然氨基酸掺入的程度）进行评估。我们建议，第三个参数的基板识别，percent，是同样重要的。许可UAARS。其宽松的底物识别特性允许它们掺入多个非天然氨基酸（但不是天然氨基酸），将消除为许多新的UAA产生新的UaaRS的需要。在这里，我们概述了快速，轻松地评估现有UaaRS的许可和生成，许可UaaRS的方法。在原理实验的证明中，我们确定了两个UaaRS的结构相关的氟化UAA（F-19-UAA）的家庭的perception的程度。然后，我们增加了初始UaaRS的概率，以允许纳入F-19-UAA家族。最后，我们验证了这些新的F-19-UAA作为探针的蛋白质结构和动力学的氟NMR研究的效用。我们希望这项工作的结果将增加UAA技术的可及性和新UAA在蛋白质中的使用。
Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. Using these techniques, researchers can precisely control the position and number of novel chemical moieties in a protein, via introducing the novel R group of UAAs, that are genetically encoded in the protein's primary structure. The substrate recognition properties of a natural aminoacyl-tRNA synthetase (aaRS) must be modified in order to incorporate UAAs into proteins. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibility of this important technology. At present, engineered unnatural aminoacyl-tRNA synthetases (UaaRS) are evaluated on their translational efficiency (the extent to which they allow for incorporation or UAAs into protein) and fidelity (the extent to which they prevent incorporation of natural amino acids). We propose that a third parameter of substrate recognition, permissivity, is equally important. Permissive UaaRSs. Whose relaxed Substrate recognition properties allow them to incorporate multiple unnatural amino acids (but not natural amino acids), Would eliminate the need to generate new UaaRSs for many new, UAAs. Here, we outline methods for quickly and easily assessing the permissivity of existing UaaRSs and for generating, permissive UaaRSs. In proof of principle experiments, we determined the degree of permissivity of two UaaRSs for a family of structurally related fluorinated UAAs (F-19-UAAs). We then increased the permissivity of the initial UaaRSs to allow for incorporation of the family of F-19-UAAs. Finally, we validated the utility of these new F-19-UAAs as probes for fluorine NMR studies of protein structure and dynamics. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins.