Genetic Encoding of 7-Aza-l-tryptophan: Isoelectronic Substitution of a Single CH-Group in a Protein for a Nitrogen Atom for Site-Selective Isotope Labeling

ACS Sens. 2023 Nov 24;8(11):4402-4406. doi: 10.1021/acssensors.3c01904. Epub 2023 Oct 27.

Abstract

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 β-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from 15N/13C-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. 15N-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with 15N/13C-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.

Keywords: 7-azatryptophan; NMR spectroscopy; genetic encoding; isoelectronic substitution; selective isotope labeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids
  • Humans
  • Isotope Labeling
  • Nitrogen
  • Serine
  • Tryptophan
  • Zika Virus Infection*
  • Zika Virus*

Substances

  • Tryptophan
  • Nitrogen
  • Amino Acids
  • Serine