Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.
Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog.
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DOI：
10.1093/nar/gkv1255
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Guo LT
中科院分区：
文献类型：
--
作者：
Englert M;Nakamura A;Wang YS;Eiler D;Söll D;Guo LT
基因编码的非规范氨基酸是蛋白质研究和工程的有力工具；特别是，它们允许取代感兴趣的蛋白质中的单个化学基团或原子。其中一种氨基酸是色氨酸（Trp）类似物3-苯并噻吩-l-丙氨酸（Bta），在五元环上具有亚胺-硫取代。与色氨酸不同，Bta不能形成氢键，但保留了色氨酸残基的其他性质。在这里，我们提出了一种pyrolyyl - trna合成酶衍生的工程酶BtaRS，它能够有效地和位点特异性地将Bta结合到体内感兴趣的蛋白质中。此外，我们报告了BtaRS•Bta复合物的2.1 Å-resolution晶体结构，以显示BtaRS如何区分Bta与典型氨基酸，包括色氨酸。为了显示其在蛋白质诱变中的效用，我们使用Bta来取代金黄色葡萄球菌硫氧还蛋白活性位点的Trp28残基。本实验表明，对活性位点维持起重要作用的不是Trp28和Asp58之间的氢键，而是Trp28粗大的芳香侧链。总的来说，我们的研究为检查蛋白质中色氨酸的功能提供了一个新的和强大的工具。
Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.