Genetic Incorporation of Twelve meta-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant

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• 作者：Yane-Shih Wang ; Xinqiang Fang ; Hsueh-Ying Chen ; Bo Wu ; Zhiyong U. Wang ; Christian Hilty ; Wenshe R. Liu
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：February 15, 2013
• 年：2013
• 卷：8
• 期：2
• 页码：405-415
• 全文大小：467K
• 年卷期：v.8,no.2(February 15, 2013)
• ISSN：1554-8937

文摘

When coexpressed with its cognate amber suppressing tRNA_CUA^Pyl, a pyrrolysyl-tRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro, ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive ¹⁹F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone, alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.