期刊
ACS SENSORS
卷 -, 期 -, 页码 -出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.3c01904
关键词
7-azatryptophan; genetic encoding; isoelectronicsubstitution; NMR spectroscopy; selective isotopelabeling
Genetic encoding of noncanonical amino acids allows high-yielding site-selective isotope labeling in vivo, which has important applications in protein research, as demonstrated by the successful production of mutants of Zika virus protease.
Genetic encoding of a noncanonical amino acid (ncAA) in an in vivo expression system requires an aminoacyl-tRNA synthetase that specifically recognizes the ncAA, while the ncAA must not be recognized by the canonical protein expression machinery. We succeeded in genetically encoding 7-aza-tryptophan (7AW), which is isoelectronic with tryptophan. The system is fully orthogonal to protein expression in Escherichia coli, enabling high-yielding site-selective isotope labeling in vivo. 7AW is readily synthesized from serine and 7-aza-indole using a tryptophan synthetase beta-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from N-15/C-13-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. N-15-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with N-15/C-13-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.
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