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

Abstract

Ni²⁺ affinity columns are widely used for protein purification, but they carry the risk that Ni²⁺ 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 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 Ni²⁺ coordinated to amino acid side chains and [Ni(EDTA)(H₂O)_n]²⁻. Dialysis against ≥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, the binding of Ni²⁺ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni²⁺.