Chemistry of Periodate-Mediated Cross-Linking of 3,4-Dihydroxylphenylalanine-Containing Molecules to Proteins

Abstract

Chemical cross-linking is an attractive approach to map peptide−protein and protein−protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation (Burdine, L.; Gillette, T. G.; Lin, H.-J.; Kodadek, T. J.Am.Chem.Soc. 2004, 126, 11442−11443). We report here a study on the chemistry of DOPA−protein cross-linking. First, using a peptide nucleic acid templated system, we identified the α-amino, ε-amino of Lys, imidazole of His, and thiol of Cys as functional groups capable of attacking DOPA ortho-quinone. Second, we demonstrated that periodate-induced DOPA−protein cross-linking could be carried out efficiently at neutral pH in the presence of excess aliphatic 1,2-diols such as ethylene glycol, lactose, and adenosine triphosphate. This result indicated that DOPA−protein cross-linking and 1,2-diol oxidative cleavage proceed via different mechanisms and that carbohydrates will not interfere with this process when carried out in crude cell extracts or on intact cells.