KLEANTHOUS, COLIN, VAN DEN BREMER, EWALD T.J., JISKOOT, WIM et al. (4 more authors) (2002) Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry. Protein Science. pp. 1738-1752. ISSN 1469-896X
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 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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | Copyright © 2002 The Protein Society. |
Keywords: | Colicin E9,metal ion binding,protein conformation,hydrogen exchange,mass spectrometry |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Christina Hudson |
Date Deposited: | 20 Feb 2006 |
Last Modified: | 16 Oct 2024 11:56 |
Published Version: | https://doi.org/10.1110/ps.0200502 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1110/ps.0200502 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:1023 |