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用于蛋白质结构解析的魔角旋转固体NMR PCS方法

(J. Am. Chem. Soc., 2013, 135, 8294–8303)

   

  魔角旋转固体NMR是获得具有原子分辨率的三维结构的独特技术。它能够研究那些因难以结晶而不易被X-ray解析或者因分子量太大而不易被液体NMR解析的生物大分子。目前,固体NMR主要通过偶极—偶极耦合获取长程距离约束条件——该方法难以快速测量足够多的长程距离条件,从而降低了固体NMR解析蛋白质结构的效率。本研究表明:固体NMR利用伪接触位移(Pseudocontact shift, PCS)Rosetta计算方法,同样可以解析高分辨率的蛋白质结构,而不需要传统的基于偶极耦合的距离测量方法。以GB1(56残基)为模型蛋白,我们利用探针分子4-巯基甲基吡啶二羧酸(4-mercaptomethyl-dipicolinic acid, 4MMDPA)在蛋白质的不同位点上引入具有PCS效应的顺磁金属离子,进而获得了一系列的PCS值。根据这些PCS,我们得到了可信的(与X-ray结构对比)、长达20埃的长程距离。此外,我们还证实:在蛋白质的不同位点引入磁化率不同的顺磁离子,可以覆盖蛋白质的不同区域,从而极大地增加长程PCS距离的数量。最终,结合固体NMR PCS距离和Rosetta方法计算得到了RMSD0.7GB1蛋白质结构(相对于X-ray晶体结构)。

 

 

Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

Abstract     Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar–dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ~20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure. 

 








 
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