Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells,ACS Chemical Biology

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Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2021-09-30 , DOI: 10.1021/acschembio.1c00649
Riley M Bednar ₁ , Subhashis Jana ₁ , Sahiti Kuppa ₂ , Rachel Franklin ₁ , Joseph Beckman ₁ , Edwin Antony ₂ , Richard B Cooley ₁ , Ryan A Mehl ₁

Affiliation

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically defined protein–protein cross-linking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

中文翻译：

两种相互正交的生物正交氨基酸的遗传结合，可在细胞中实现高效的蛋白质双标记

在体内多个位点进行位点特异性修饰蛋白质的能力将能够以前所未有的详细程度研究其天然环境中的蛋白质功能。在这里，我们提出了一种通用的两步策略来实现这一目标，包括将两种不同的非经典氨基酸进行位点特异性编码，这些氨基酸带有生物正交手柄到体内的蛋白质中，然后是相互正交的标记。这种通用方法，我们称之为双重编码和labeling (DEAL)，使我们能够有效地将含有四嗪和叠氮的氨基酸编码到蛋白质中，并首次证明，当位点特异性时，具有应变烯烃和炔烃标记的生物正交标记反应可以同时在细胞内以高产率发挥作用编码在单一蛋白质中。使用我们的 DEAL 系统，我们能够在活的大肠杆菌细胞内进行拓扑定义的蛋白质-蛋白质交联、分子内钉合和荧光团的位点特异性安装，以及研究酵母复制蛋白 A 的 DNA 结合特性体外。通过在体内对蛋白质进行有效的双重修饰, 这种 DEAL 方法为体内蛋白质的表征和工程提供了一种工具。

更新日期：2021-11-19

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