Structural basis of improved second-generation 3-nitro-tyrosine tRNA synthetases.
Structural basis of improved second-generation 3-nitro-tyrosine tRNA synthetases.
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DOI：
10.1021/bi5001239
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发表时间：
2014-04-01
期刊：
影响因子：
2.9
通讯作者：
Mehl RA
中科院分区：
文献类型：
--
作者：
Cooley RB;Feldman JL;Driggers CM;Bundy TA;Stokes AL;Karplus PA;Mehl RA
遗传密码扩增提供了将大量非典型氨基酸（ncAA）位点特异性地掺入蛋白质中以用于多种应用的能力，但是低ncAA掺入效率会阻碍这种强大技术的实用性。当研究含有翻译后修饰3-硝基-酪氨酸（nitroTyr）的蛋白质时，我们开发了第二代氨基酰基tRNA合成酶（RS），其以比先前报道的效率大约高一个数量级的效率掺入硝基Tyr，并且提高了我们阐明升高的细胞硝基Tyr水平在人类疾病中的作用的能力（例如，佛朗哥，M。等，）。在这里，我们将探讨这些第二代RS所取得的改进的起源。这些合成酶中最有效的晶体结构揭示了在第二代硝基Tyr-RS中观察到的增强效率的分子基础。尽管当表达培养基补充有ImM硝基Tyr时，Tyr未可检测地掺入蛋白质中，但是第一代和第二代RS之间的主要差异在于第二代RS具有与Tyr结合更相容的活性位点。第二代硝基Tyr-RS的这一特征似乎是从突变体文库中选择时使用不太严格的标准的结果。对相同突变体文库进行的不同选择策略产生效率显著提高的硝基Tyr-RS的观察结果表明，对已建立的选择方案进行优化可以显著提高ncAA-RS效率，从而提高该技术的整体效用。
Genetic code expansion has provided the ability to site-specifically incorporate a multitude of noncanonical amino acids (ncAAs) into proteins for a wide variety of applications, but low ncAA incorporation efficiency can hamper the utility of this powerful technology. When investigating proteins containing the post-translational modification 3-nitro-tyrosine (nitroTyr), we developed second-generation amino-acyl tRNA synthetases (RS) that incorporate nitroTyr at efficiencies roughly an order of magnitude greater than those previously reported and that advanced our ability to elucidate the role of elevated cellular nitroTyr levels in human disease (e.g., Franco, M. et al. , , ). Here, we explore the origins of the improvement achieved in these second-generation RSs. Crystal structures of the most efficient of these synthetases reveal the molecular basis for the enhanced efficiencies observed in the second-generation nitroTyr-RSs. Although Tyr is not detectably incorporated into proteins when expression media is supplemented with 1 mM nitroTyr, a major difference between the first- and second-generation RSs is that the second-generation RSs have an active site more compatible with Tyr binding. This feature of the second-generation nitroTyr-RSs appears to be the result of using less stringent criteria when selecting from a library of mutants. The observation that a different selection strategy performed on the same library of mutants produced nitroTyr-RSs with dramatically improved efficiencies suggests the optimization of established selection protocols could lead to notable improvements in ncAA-RS efficiencies and thus the overall utility of this technology.
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影响因子：
56.9
作者：
Choudhary, Chunaram;Kumar, Chanchal;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
2.9
作者：
Miyake-Stoner, Shigeki J.;Refakis, Christian A.;Mehl, Ryan A.
通讯作者：
Mehl, Ryan A.
影响因子：
3.5
作者：
Heinemann IU;Rovner AJ;Aerni HR;Rogulina S;Cheng L;Olds W;Fischer JT;Söll D;Isaacs FJ;Rinehart J
通讯作者：
Rinehart J
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
15
作者：
Turner, JM;Graziano, J;Schultz, PG
通讯作者：
Schultz, PG