Rapid Measurement of Pseudocontact Shifts in Metalloproteins by Proton-Detected Solid-State NMR Spectroscopy
Rapid Measurement of Pseudocontact Shifts in Metalloproteins by Proton-Detected Solid-State NMR Spectroscopy
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DOI：
10.1021/ja306813j
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发表时间：
2012-09-12
影响因子：
15
通讯作者：
Pintacuda, Guido
中科院分区：
文献类型：
--
作者：
Knight, Michael J.;Felli, Isabella C.;Pintacuda, Guido
赝接触位移（PCSs）出现在磁化率张量各向异性的顺磁体系中。PCSs取决于距顺磁中心的距离以及相对于磁化率张量的位置，并且它们可在蛋白质结构测定中用作结构约束。我们表明，使用¹H检测的固态相关能够在一种大型金属蛋白——钴（II）取代的超氧化物歧化酶（Co²⁺ - SOD）中方便且快速地检测和指认位点特异性PCSs，包括已分辨的¹H PCSs。仅用3毫克样品和少量实验，就测量并指认了数百个PCSs，随后将这些PCSs与¹H - ¹H距离和二面角约束相结合，以确定蛋白质主链的几何结构，其精度可与抗磁蛋白质的最先进液态测定相媲美，包括一个明确的活性位点。
Pseudocontact shifts (PCSs) arise in para-magnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of H-1-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved H-1 PCSs, in a large metalloprotein, Co2+-substituted superoxide dismutase (Co2+-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with H-1-H-1 distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.