Hostname: page-component-7bb8b95d7b-w7rtg Total loading time: 0 Render date: 2024-10-06T17:53:39.088Z Has data issue: false hasContentIssue false

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

Published online by Cambridge University Press:  01 August 1999

JONATHAN P. HANNAN
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
SARA B.-M. WHITTAKER
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
SHARON L. DAVY
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
ULRIKE C. KÜHLMANN
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
ANSGAR J. POMMER
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
ANDREW M. HEMMINGS
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom Schools of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
RICHARD JAMES
Affiliation:
Schools of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
COLIN KLEANTHOUS
Affiliation:
Schools of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
GEOFFREY R. MOORE
Affiliation:
School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
Get access

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+.

Type
FOR THE RECORD
Copyright
© 1999 The Protein Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)