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

When coexpressed with its cognate amber suppressing tRNACUA 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 non...

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Published in:ACS chemical biology 2013-02, Vol.8 (2), p.405-415
Main Authors: Wang, Yane-Shih, Fang, Xinqiang, Chen, Hsueh-Ying, Wu, Bo, Wang, Zhiyong U, Hilty, Christian, Liu, Wenshe R
Format: Article
Language:English
Subjects:
Amino Acyl-tRNA Synthetases - genetics
Amino Acyl-tRNA Synthetases - metabolism
Escherichia coli - genetics
Lysine - analogs & derivatives
Lysine - genetics
Lysine - metabolism
Models, Molecular
Molecular Structure
Mutant Proteins - genetics
Mutant Proteins - metabolism
Mutation
Phenylalanine - analogs & derivatives
Phenylalanine - chemistry
Phenylalanine - genetics
Substrate Specificity
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Summary:When coexpressed with its cognate amber suppressing tRNACUA 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.
ISSN:1554-8929
1554-8937
DOI:10.1021/cb300512r