NMR study of Ni 2+ binding to the H‐N‐H endonuclease domain of colicin E9

Ni 2 + affinity columns are widely used for protein purification, but they carry the risk that Ni 2 + ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni 2 + acquired from Ni 2 + affinity columns, and appears to bind [Ni(EDTA)(H 2 O) n ] 2− at low ionic strength. NMR was used to detect the presence of both Ni 2 + coordinated to amino acid side chains and [Ni(EDTA)(H 2 O) n ] 2− . Dialysis against ≥0.2 MNaCl was required to remove the [Ni(EDTA)(H 2 O) n ] 2− . The NMR procedure we have used to characterize the presence of Ni 2 + and [Ni (EDTA)(H 2 O) n ] 2− should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni 2 + seems likely to be physiologically relevant, and the NMR data complement recent X‐ray crystallographic evidence concerning the number of histidine ligands to bound Ni 2 +.