Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine

J Biochem. 2007 Mar;141(3):335-43. doi: 10.1093/jb/mvm036. Epub 2007 Jan 3.

Abstract

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Azides / chemistry*
  • Biotinylation / methods
  • Calmodulin / chemistry*
  • Cell-Free System
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Organophosphorus Compounds / chemistry
  • Protein Processing, Post-Translational / physiology*
  • RNA, Transfer, Tyr / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine-tRNA Ligase / metabolism

Substances

  • Azides
  • Calmodulin
  • Organophosphorus Compounds
  • RNA, Transfer, Tyr
  • 3-azidotyrosine
  • triphenylphosphine
  • Tyrosine
  • Tyrosine-tRNA Ligase