Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry.
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.
van den Bremer
Ewald T J
ET
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Jiskoot
Wim
W
James
Richard
R
Moore
Geoffrey R
GR
Kleanthous
Colin
C
Heck
Albert J R
AJ
Maier
Claudia S
CS
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
D001426:Bacterial Proteins; D003087:Colicins; D007477:Ions; D008670:Metals; C007104:colicin immunity proteins; D014364:Tryptophan; D003851:Deoxyribonucleases; D015032:Zinc