An engineered aryl azide ligase for site-specific mapping of protein-protein interactions through photo-cross-linking

Angew Chem Int Ed Engl. 2008;47(37):7018-21. doi: 10.1002/anie.200802088.

Abstract

We re-engineered the small-molecule binding site of E. coli lipoic acid ligase (LplA) to accept a fluorinated aryl azide probe in place of lipoic acid. The mutated ligase can covalently attach the aryl azide to recombinant proteins fused to a 17-amino acid recognition sequence for LplA (called "LAP"). Labeling is highly specific, modifying LAP fusion proteins to the exclusion of all endogenous mammalian proteins. In cell lysate, we labeled FKBP with aryl azide and demonstrated rapamycin-dependent photocrosslinking to FRB.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Azides / chemistry*
  • Binding Sites
  • Cell Line
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • HeLa Cells
  • Humans
  • Ligases / chemistry*
  • Ligases / genetics
  • Molecular Probes / chemistry*
  • Mutation
  • Peptides / chemistry
  • Photochemistry
  • Protein Engineering*
  • Protein Interaction Mapping / methods*
  • Thioctic Acid / chemistry
  • Ultraviolet Rays

Substances

  • Azides
  • Escherichia coli Proteins
  • Molecular Probes
  • Peptides
  • lplA protein, E coli
  • Thioctic Acid
  • Adenosine Triphosphate
  • Ligases