Genetic Incorporation
of Twelve <i>meta</i>-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant
Yane-Shih Wang
Xinqiang Fang
Hsueh-Ying Chen
Bo Wu
Zhiyong U. Wang
Christian Hilty
Wenshe R. Liu
10.1021/cb300512r.s001
https://acs.figshare.com/articles/journal_contribution/Genetic_Incorporation_of_Twelve_i_meta_i_Substituted_Phenylalanine_Derivatives_Using_a_Single_Pyrrolysyl_tRNA_Synthetase_Mutant/2442646
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.
2013-02-15 00:00:00
346A
protein
19 F NMR probe
azide moieties