Genetic Incorporation of Twelve <i>meta</i>-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant
2013-02-15T00:00:00Z (GMT) by
When coexpressed with its cognate amber suppressing tRNA<sub>CUA</sub><sup>Pyl</sup>, a pyrrolysyl-tRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 <i>meta</i>-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in <i>Escherichia coli</i>. 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 <sup>19</sup>F 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.
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