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Synthetase polyspecificity as a tool to modulate protein function.

Douglas D Young1, Steffen Jockush, Nicholas J Turro

  • 1Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

Bioorganic & Medicinal Chemistry Letters
|November 2, 2011
PubMed
Summary
This summary is machine-generated.

Researchers engineered bacterial enzymes (aminoacyl-tRNA synthetases) to incorporate multiple unnatural amino acids (UAAs) into proteins. This discovery expands the toolkit for protein engineering and modifying GFP

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Site-specific incorporation of unnatural amino acids (UAAs) into proteins relies on engineered aminoacyl-tRNA synthetases (aaRSs).
  • Previously evolved aaRSs sometimes exhibit polyspecificity, recognizing multiple UAAs.
  • This characteristic can be leveraged for broader applications in protein engineering.

Purpose of the Study:

  • To investigate the polyspecificity of an aaRS evolved for a coumarin-containing amino acid.
  • To utilize this polyspecificity for introducing diverse UAAs into Green Fluorescent Protein (GFP).
  • To explore the impact of UAA incorporation on GFP's photophysical properties.

Main Methods:

  • Evolution of specific aminoacyl-tRNA synthetases (aaRSs).
  • Characterization of aaRS polyspecificity using various unnatural amino acids.
  • Site-specific incorporation of UAAs into the GFP fluorophore in bacteria.
  • Analysis of altered photophysical properties of modified GFP.

Main Results:

  • An aaRS evolved for a specific coumarin UAA demonstrated significant polyspecificity.
  • Multiple UAAs were successfully incorporated into the GFP fluorophore using this polyspecific aaRS.
  • The photophysical properties of GFP were demonstrably altered by the introduced UAAs.

Conclusions:

  • Engineered aaRSs can exhibit useful polyspecificity for introducing multiple UAAs.
  • This approach enables the facile modification of protein fluorophores like GFP.
  • Polyspecific aaRSs offer a versatile platform for expanding the functional capabilities of proteins.