A critical role for tyrosine residues in His6Ni-mediated protein cross-linking.
A new type of affinity cross-linking strategy has been developed in which His6-tagged proteins can be cross-linked to their binding partners in the presence of unmodified proteins (D. Fancy, K. Melcher, S. A. Johnston, and T. Kodadek, 1996, Chem. Biol. 3, 551-559). The chemistry involves the addition of Ni(II) to the His6 tag, followed by oxidation of the metal with a peracid. It is shown here that, in addition to the His6 tag, a tyrosine residue placed in close proximity to the metal-binding site can strongly stimulate the yield of cross-linked product. This finding has important practical implications in the use of the His6-Ni-based cross-linking reaction for the analysis of multiprotein complexes.
Fancy
D A
DA
Center for Biomedical Inventions, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas, 75235-8573, USA.
Kodadek
T
T
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D009532:Nickel / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D003432:Cross-Linking Reagents; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D006639:Histidine; D009532:Nickel