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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
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A genetically encoded acrylamide functionality.

Yan-Jiun Lee1, Bo Wu, Jeffrey E Raymond

  • 1Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

ACS Chemical Biology
|June 6, 2013
PubMed
Summary
This summary is machine-generated.

Researchers genetically encoded Nε-Acryloyl-l-lysine in E. coli for versatile protein modifications. This noncanonical amino acid enables unique reactions like hydrogel formation and fluorescent labeling in vitro and in vivo.

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Area of Science:

  • Biochemistry
  • Synthetic Biology
  • Chemical Biology

Background:

  • Noncanonical amino acids offer unique chemical functionalities for protein engineering.
  • Genetic encoding systems expand the toolbox for incorporating unnatural amino acids into proteins.
  • Nε-Acryloyl-l-lysine presents a reactive acrylamide moiety for diverse chemical transformations.

Purpose of the Study:

  • To genetically encode Nε-Acryloyl-l-lysine in Escherichia coli.
  • To explore the reactivity of the incorporated Nε-Acryloyl-l-lysine for protein modification.
  • To demonstrate site-specific protein labeling and hydrogel formation using this unnatural amino acid.

Main Methods:

  • Utilized a pyrrolysyl-tRNA synthetase mutant and tRNACUAPyl for genetic encoding of Nε-Acryloyl-l-lysine.
  • Investigated 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition reactions.
  • Performed protein modification with thiol-containing nucleophiles and radical copolymerization.
  • Demonstrated site-specific fluorescent labeling via 1,3-dipolar cycloaddition in vitro and in living cells.

Main Results:

  • Successfully achieved genetic incorporation of Nε-Acryloyl-l-lysine in E. coli.
  • Demonstrated efficient protein modification with thiols under mild conditions.
  • Showcased rapid protein incorporation into polyacrylamide hydrogels at physiological pH.
  • Achieved site-specific, turn-on fluorescent labeling of proteins in vitro and in living cells.

Conclusions:

  • Nε-Acryloyl-l-lysine is a versatile noncanonical amino acid for genetically encoded protein modification.
  • The acrylamide moiety enables diverse bioorthogonal reactions, including hydrogel formation and fluorescent labeling.
  • This technology provides a powerful platform for creating specifically modified proteins for various applications.