Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein

Biochemistry. 1998 Mar 31;37(13):4397-406. doi: 10.1021/bi9728046.


The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetates / chemistry
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Cross-Linking Reagents / chemistry*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins*
  • Nickel / chemistry
  • Oligopeptides / biosynthesis
  • Oligopeptides / chemistry*
  • Oligopeptides / genetics
  • Oxidants / chemistry
  • Oxidation-Reduction
  • Periplasmic Proteins*
  • Phthalic Acids / chemistry
  • Point Mutation
  • Protein Conformation
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / chemistry
  • Tyrosine / chemistry
  • Tyrosine / genetics


  • Acetates
  • Bacterial Proteins
  • Cross-Linking Reagents
  • Eco protein, E coli
  • Escherichia coli Proteins
  • Oligopeptides
  • Oxidants
  • Periplasmic Proteins
  • Phthalic Acids
  • Recombinant Fusion Proteins
  • Tyrosine
  • diglycyl-histidine
  • Nickel
  • monoperoxyphthalate
  • Trypsin