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通过胺氨基酸-tRNA结合酶的基质识别

Josseline Ramos-Figueroa^1,2, Haoqian Liang³, Wilfred A van der Donk^1,2,3

¹Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

Proceedings of the National Academy of Sciences of the United States of America

|March 19, 2025

概括

新的胺氨基-tRNA连接酶 (PEARLs) 可以将氨基酸添加到中. 研究人员确定了一种添加托的PeARL,发现它具有最小的序列要求,可以用于蛋白质标签.

科学领域:

生物化学生物化学
分子生物学分子生物学
酶学是一种酶学.

背景情况:

胺氨基酸-tRNA连接酶 (PEARLs) 是一种酶,它将氨基酸连接到C末端.
PEARLs对基质和氨基tRNA的识别机制尚不清楚.
以前的研究已经确定了能够结合各种氨基酸的PEARLs,包括Cys,Ala,Trp,Gly,Leu,Asn和Thr.

研究的目的:

为了研究一种特定的PEARL酶的基质特异性,该酶负责托的结合.
为了阐明最小的序列要求,通过这个PEARL.识别基质.
探索这个PEARL作为C端蛋白标记工具的潜力.

主要方法:

利用细胞自由表达 (CFE) 快速测定基质变体.
进行了切断和替换实验,以确定最小序列要求.
采用AlphaFold3建模来预测绑定相互作用.
进行了位点导向的突变发生,以验证tRNA,ATP和Mg2+的预测结合模式.
应用已识别的最小序列作为C端标签的蛋白质标签.

主要成果:

研究的PEARL对其基质C端氨基酸的变化表现出了显著的耐受性.
确定了用于基质识别的最小序列要求.
阿尔法Fold3建模提供了对基质,tRNA,ATP和Mg2+结合的见解.
突变性实验支持保存残留在识别tRNA3'-CCA序列中的作用.
该酶成功地被用于用托和5--托对eGFP,lyszyme和MBP进行C端标记.

结论:

这些发现揭示了和tRNA识别的关键方面,通过一个含托的PEARL.
该研究确定了基质的最小序列要求,促进了酶工程.
开发的基于PEARL的蛋白质标签系统为特定位置的C端修改提供了一种新的方法.

关键词:

氨基酸-tRNA的氨基酸.结合方式结合方式键是一种键.

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