A Genetically Encoded Acrylamide Functionality

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• 作者：Yan-Jiun Lee ; Bo Wu ; Jeffrey E. Raymond ; Yu Zeng ; Xinqiang Fang ; Karen L. Wooley ; Wenshe R. Liu
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：August 16, 2013
• 年：2013
• 卷：8
• 期：8
• 页码：1664-1670
• 全文大小：389K
• 年卷期：v.8,no.8(August 16, 2013)
• ISSN：1554-8937

文摘

N^蔚-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNA_CUA^Pyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with N^蔚-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with N^蔚-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.