Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase.
Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase.
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完成电路：直接观察 13C,15N 双量子光谱允许在乙酰还原酮双加氧酶的顺磁中心附近进行连续共振分配。
DOI：
10.1021/ja710187x
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发表时间：
2008
影响因子：
15
通讯作者：
Pochapsky,ThomasC
中科院分区：
文献类型：
--
作者：
Pochapsky,SusanSondej;Sunshine,JoelC;Pochapsky,ThomasC
酸性还原内酯双加氧酶(ARD)是一种由179个残基组成的酶，活性部位含有顺磁性的镍离子。由于电子与核自旋的相互作用，Ni+2的−∼9ä中的1H共振被展宽到无法检测到的程度。因此，1H检测的多维核磁共振实验不适合ARD活性中心的结构表征，也没有等结构的抗磁同系物可用。快速循环二维直接13C检测核磁共振方法以前允许在ARD(Kostic，M.；Pochapsky，S.S.；Pochapsky，T.C.J.Am.Chem)中将(13C‘)碳与直接键合的13Cα和15N自旋相关联。Soc.2002,124,9054−9055)，而不是15N与13Cα，这是主干共振顺序分配的关键连接。现已证明，完全样品氚与使用冷探针/前置放大器的直接13C探测相结合，允许通过四脉冲双量子实验CAN进行单键13Cα−15N关联。结合其他13C直接观察2D核磁共振实验的数据，CAN数据允许对靠近活性中心金属的ARD中的许多共振进行顺序指定。
Acireductone dioxygenase (ARD) is a 179-residue enzyme containing a paramagnetic Ni+2ion in the active site. Because of electron−nuclear spin interactions,1H resonances within ∼9 Å of the Ni+2are 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 direct13C detection NMR methods previously allowed correlation of carbonyl (13C‘) carbons with directly bonded13Cαand15N spins in ARD (Kostic, M.; Pochapsky, S. S.; Pochapsky, T. C.J. Am.Chem. Soc.2002,124, 9054−9055), but not15N with13Cα, a critical connection for sequential assignment of backbone resonances. It is now shown that complete sample deuteration combined with direct13C detection using a cold probe/preamplifier permits the one-bond13Cα−15N correlation to be made via a four-pulse double-quantum experiment CAN. Combined with data from other13C direct-observe 2D NMR experiments, CAN data permits sequential assignments to be made for many resonances in ARD close to the active-site metal.