Determining protein-protein interactions by oxidative cross-linking of a glycine-glycine-histidine fusion protein.
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.
Brown
K C
KC
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94131, USA.
Yu
Z
Z
Burlingame
A L
AL
Craik
C S
CS
D000085:Acetates / Q000737:chemistry; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins; D010795:Phthalic Acids / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D000085:Acetates; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D016877:Oxidants; D033903:Periplasmic Proteins; D010795:Phthalic Acids; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; C037026:diglycyl-histidine; D009532:Nickel; C063907:monoperoxyphthalate; D014357:Trypsin