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See detailPositively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 beta-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis
Jacquin, Olivier ULg; Balbeur, Dorothée ULg; Damblon, Christian ULg et al

in Journal of Molecular Biology (2009), 392(5), 1278-1291

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum ... [more ▼]

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase H from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K-1/K-2 >= 5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K-2 < 80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility. (C) 2009 Elsevier Ltd. All rights reserved. [less ▲]

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See detailElectron detachment dissociation (EDD) pathways in oligonucleotides
Kinet, Catherine ULg; Gabelica, Valérie ULg; Balbeur, Dorothée ULg et al

in International Journal of Mass Spectrometry (2009), 283

Electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) are two novel fragmentation methods yielding radicals from negatively charged ions. With the goal of comparing EDD ... [more ▼]

Electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) are two novel fragmentation methods yielding radicals from negatively charged ions. With the goal of comparing EDD, EPD and the more traditional Collision-Induced Dissociation (CID) and Infrared Multiphoton Disscociation (IRMPD) fragmentation processes in oligonucleotides, we studied here the EDD fragmentation pathways of oligonucleotides of varying length. We chose polythymine oligonucleotides because these are the least prone to secondary structure formation, and found complete sequence coverage by EDD for up to dT20. We also found that the fragmentation pathways change with oligonucleotide length: electron detachment is a mandatory step in the fragmentation of larger sequences, while shorter oligonucleotides can also fragment via direct electronic or vibrational excitation by the electrons. This is supported by (1) the fact that continuous ejection of the charge reduced species does not totally prevent fragmentation of short oligonucleotides dT5 and dT6, (2) the fact that CID and EDD fragments are more similar for small oligonucleotides (although double resonance experiments show that they are not all issued from the same mechanisms), and (3) the fact that electron-induced dissociation (EID) of singly charged dT3 and dT4 gives similar fragments as EDD of doubly charged dT5 and dT6. Finally, the detachment efficiency as a function of the nature of the nucleobase was studied. The effect of base on electron detachment in EDD (G > T > A > C) is different than in EPD (G > A > C > T), indicating different electron loss mechanisms. [less ▲]

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See detailDetection of Oligonucleotide Gas-Phase Conformers: H/D Exchange and Ion Mobility as Complementary Techniques
Balbeur, Dorothée ULg; Widart, Joëlle ULg; Leyh, Bernard ULg et al

in Journal of the American Society for Mass Spectrometry (2008), 19(7), 938-946

Gas-phase hydrogen/deuterium exchange of small oligonucleotides (dTG, dC6 and C6) with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer ... [more ▼]

Gas-phase hydrogen/deuterium exchange of small oligonucleotides (dTG, dC6 and C6) with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. Ion activation experiments were conducted by accelerating the ions at the entrance of the H/D exchange cell under conditions promoting exclusively collisional isomerization. These experiments allowed us to assess the presence of several conformers, and to probe the height of the isomerization barrier separating these conformers. Ion mobility experiments were also performed. Their results were consistent with the H/D exchange data. A model accounting for the competing isomerization and H/D exchange reactions is proposed. Comparing the ion acceleration experiments for H/D exchange and for ion mobility reveals that the most compact conformer displays the fastest H/D exchange. This observation shows that H/D exchange and ion mobility provide us with complementary information because hydrogen accessibility and macromolecule compactness are not univocally associated. [less ▲]

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See detailConformationally driven gas-phase H/D exchange of dinucleotide negative ions
Balbeur, Dorothée ULg; Dehareng, Dominique ULg; De Pauw, Edwin ULg

in Journal of the American Society for Mass Spectrometry (2007), 18(10), 1827-1834

Gas-phase hydrogen/deuterium exchange of six deprotonated dinucleoticles with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. To ... [more ▼]

Gas-phase hydrogen/deuterium exchange of six deprotonated dinucleoticles with CD3OD was performed in the second hexapole of a Fourier transform ion-cyclotron resonance (FTICR) mass spectrometer. To complete these experiments, dynamic simulations were carried out to investigate the different conformations adopted by the dinucleotides. In the experimental conditions and in integrating the experimental and theoretical results, H/D exchange was shown to be controlled by hydrogen accessibility and not by the chemical nature of the heteroatom bearing the exchangeable hydrogen. A model including simultaneous H/D exchanges at the experimental time scale was used to reproduce the dinucleotide H/D exchange kinetic plots. The relay mechanism was not relevant for dinucleotides. This allowed the H/D exchange rates to be directly linked to conformations. [less ▲]

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