KLEANTHOUS, COLIN, VAN DEN BREMER, EWALD T.J., JISKOOT, WIM, James, Richard, Moore, Geoffrey R., HECK, ALBERT J.R. and MAIER, CLAUDIA S. (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 1469-896XFull text available as:
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.
|Copyright, Publisher and Additional Information:||Copyright © 2002 The Protein Society.|
|Keywords:||Colicin E9, metal ion binding, protein conformation, hydrogen exchange, mass spectrometry|
|Academic Units:||The University of York > Biology (York)|
|Depositing User:||Christina Hudson|
|Date Deposited:||20 Feb 2006|
|Last Modified:||17 Oct 2013 14:32|