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 His(6)-tagged proteins can be crosslinked 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 His(6) tag, followed by oxidation of the metal with a peracid. It is shown here that, in addition to the His(6) tag, a tyrosine residue placed in close proximity to the metal-binding site can strongly stimulate the yield of crosslinked product, This finding has important practical implications in the use of the His(6)-Ni-based cross-linking reaction for the analysis of multiprotein complexes. (C) 1998 Academic Press.