Department of Biomolecular Chemistry, Institute of Molecules and Materials , Radboud University , Nijmegen , Heyendaalseweg 135 , 6525 AJ , The Netherlands.
ACS Chem Biol. 2018 Aug 17;13(8):1932-1937. doi: 10.1021/acschembio.8b00178. Epub 2018 Jun 14.
Mapping proteins at a specific subcellular location is essential to gaining detailed insight on local protein dynamics. We have developed an enzymatic strategy to label proteins on a subcellular level using arylamine N-acetyltransferase (NAT). The NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. The electron density on the aromatic ring proved important for probe activation as strong labeling was only observed with an arylhydroxamic acid bearing an electron donating substituent. We further demonstrate that, using this electron rich arylhydroxamic acid, clear labeling was achieved on a subcellular level in living cells that were transfected with a genetically targeted NAT to the nucleus or the cytosol.
对特定亚细胞位置的蛋白质进行定位对于深入了解局部蛋白质动力学至关重要。我们开发了一种使用芳基胺 N-乙酰转移酶（NAT）在亚细胞水平上标记蛋白质的酶促策略。NAT 酶将芳基羟肟酸官能团激活成氮宾离子，该氮宾离子快速、共价且在中性条件下与邻近蛋白质的亲核残基反应。芳环上的电子密度对于探针激活很重要，因为只有带有供电子取代基的芳基羟肟酸才能观察到强烈的标记。我们进一步证明，使用这种富电子的芳基羟肟酸，可以在转染到细胞核或细胞质中具有遗传靶向 NAT 的活细胞中实现清晰的亚细胞水平标记。
