文摘
Selective chemical modification of thiol groups combined with mass spectrometry analysiswas used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is ametalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake inGram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvementof two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein.The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylationof the thiol groups monitored by quenching the reaction at different times and measuring the extent ofalkylation by mass spectrometry. Complementary experiments were carried out in the absence or presenceof EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation bythe zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by massspectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 andCys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicatesthat it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastestreacting cysteine, implying it is a surface-exposed residue.
