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Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

¹ Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
² Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
³ Division of Microbiology and Infectious Diseases, University Hospital, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom
⁴ School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
⁵ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Reprint requests to: Dr. Claudia S. Maier, Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; e-mail: c.s.maier{at}chem.uu.nl; fax: 31-30251-8219.

Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far- and near-UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid-induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn²⁺. Gas-phase dissociation experiments of the deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas-phase dissociation experiments of the ternary E9 DNase-Zn²⁺-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.

Keywords: Colicin E9; metal ion binding; protein conformation; hydrogen exchange; mass spectrometry

Abbreviations: ESI, electrospray ionization • MS, mass spectrometry • CD, circular dichroism • m/z, mass-to-charge • CsI, Cesium iodide • H/D, hydrogen/deuterium

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