title: Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry.
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.
authors.affiliation: Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
authors.forename: Ewald T J
authors.initials: ET
authors.lastname: van den Bremer
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Maier
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D003087:Colicins; D003851:Deoxyribonucleases / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D014364:Tryptophan / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D007477:Ions; D008670:Metals; C007104:colicin immunity proteins; D014364:Tryptophan; D003851:Deoxyribonucleases; D015032:Zinc