NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9

Ni2+ affinity columns are widely used for protein purification, but they ca rry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetet raacetic acid (EDTA) is normally used to remove metal ions bound adventitio usly to proteins, however, this approach does not always work. Here we repo rt that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2 + acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)(n )](2-) at low ionic strength. NMR was used to detect the presence of both N i2+ coordinated to amino acid side: chains and [Ni(EDTA)(H2O)(n)](2-). Dial ysis against greater than or equal to 0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)(n)](2-). The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)(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, th e binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the numbe r of histidine ligands to bound Ni2+.