Premium
Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time‐of‐flight mass spectrometry
Author(s) -
van den Bremer Ewald T.J.,
Jiskoot Wim,
James Richard,
Moore Geoffrey R.,
Kleanthous Colin,
Heck Albert J.R.,
Maier Claudia S.
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0200502
Subject(s) - chemistry , colicin , electrospray ionization , hydrogen–deuterium exchange , circular dichroism , mass spectrometry , metal ions in aqueous solution , metal , crystallography , chromatography , biochemistry , organic chemistry , dna , plasmid
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.