Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry |
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Authors: | van den Bremer Ewald T J Jiskoot Wim James Richard Moore Geoffrey R Kleanthous Colin Heck Albert J R Maier Claudia S |
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Affiliation: | Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. |
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Abstract: | 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 Zn(2+). 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-Zn(2+)-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability. |
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Keywords: | Colicin E9 metal ion binding protein conformation hydrogen exchange mass spectrometry |
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