Completing the Circuit: Direct-Observe 13C,15N Double-Quantum Spectroscopy Permits Sequential Resonance Assignments near a Paramagnetic Center in Acireductone Dioxygenase
详细信息 查看全文
- 作者：Susan Sondej Pochapsky ; Joel C. Sunshine ; Thomas C. Pochapsky
- 刊名：Journal of the American Chemical Society
- 出版年：2008
- 出版时间：February 20, 2008
- 年：2008
- 卷：130
- 期：7
- 页码：2156 - 2157
- 全文大小：70K
- 年卷期：v.130,no.7(February 20, 2008)
- ISSN：1520-5126
文摘
Acireductone dioxygenase (ARD) is a 179-residue enzyme containing a paramagnetic Ni+2 ion in the active site. Because of electron-nuclear spin interactions, 1H resonances within ~9 Å of the Ni+2 are broadened beyond detection. For this reason, 1H-detected multidimensional NMR experiments are not suitable for structural characterization of the active site of ARD, and no isostructural diamagnetic homologue is available. Rapid recycle two-dimensional direct 13C detection NMR methods previously allowed correlation of carbonyl (13C') carbons with directly bonded 13C
and 15N spins in ARD (Kostic, M.; Pochapsky, S. S.; Pochapsky, T. C. J. Am. Chem. Soc. 2002, 124, 9054-9055), but not 15N with 13C, a critical connection for sequential assignment of backbone resonances. It is now shown that complete sample deuteration combined with direct 13C detection using a cold probe/preamplifier permits the one-bond 13C-15N correlation to be made via a four-pulse double-quantum experiment CAN. Combined with data from other 13C direct-observe 2D NMR experiments, CAN data permits sequential assignments to be made for many resonances in ARD close to the active-site metal.
and 15N spins in ARD (Kostic, M.; Pochapsky, S. S.; Pochapsky, T. C. J. Am. Chem. Soc. 2002, 124, 9054-9055), but not 15N with 13C, a critical connection for sequential assignment of backbone resonances. It is now shown that complete sample deuteration combined with direct 13C detection using a cold probe/preamplifier permits the one-bond 13C-15N correlation to be made via a four-pulse double-quantum experiment CAN. Combined with data from other 13C direct-observe 2D NMR experiments, CAN data permits sequential assignments to be made for many resonances in ARD close to the active-site metal.