title: 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 > or =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+.
authors.affiliation: School of Chemical Sciences, University of East Anglia, Norwich, United Kingdom.
authors.forename: J P
authors.initials: JP
authors.lastname: Hannan
authors.forename: S B
authors.initials: SB
authors.lastname: Whittaker
authors.forename: S L
authors.initials: SL
authors.lastname: Davy
authors.forename: U C
authors.initials: UC
authors.lastname: Kühlmann
authors.forename: A J
authors.initials: AJ
authors.lastname: Pommer
authors.forename: A M
authors.initials: AM
authors.lastname: Hemmings
authors.forename: R
authors.initials: R
authors.lastname: James
authors.forename: C
authors.initials: C
authors.lastname: Kleanthous
authors.forename: G R
authors.initials: GR
authors.lastname: Moore
mesh_terms: D001665:Binding Sites; D003087:Colicins / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D009532:Nickel / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D003087:Colicins; D011994:Recombinant Proteins; D009532:Nickel; D004720:Endonucleases