Paramagnetic ions provide structural restraints in solid-state NMR of proteins
Paramagnetic ions provide structural restraints in solid-state NMR of proteins
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DOI：
10.1021/ja068105a
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发表时间：
2007-02-28
影响因子：
15
通讯作者：
Maletta, Massimiliano
中科院分区：
文献类型：
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作者：
Balayssac, Stephane;Bertini, Ivano;Maletta, Massimiliano
在固态（SS）中，可以获得的用于解决蛋白质的3D结构的结构限制的数量显著低于在溶液中的结构限制，并且在相对较少的情况下，它们的熟练使用实际上产生了3D结构。顺磁性金属离子，无论是作为金属蛋白中的抗磁性金属的替代物，还是插入适当设计的标签中，都被认为在溶液中提供了丰富的结构约束，其中伪接触位移是非常宝贵的。在这里，我们表明，伪接触位移（PCS）产生的顺磁性金属离子具有足够大的自旋量子数和磁化率各向异性可以很容易地测量，并在大量的，在SS光谱的蛋白质。样品是微晶钴（II）取代的基质金属蛋白酶12（MMP-12，17 kDa）。大约250个粒子被观察到的核超过20 A的金属，并发现在非常好的协议与计算。有限数量的原子核也受到晶格内相邻分子中顺磁性金属的分子间相互作用的影响，这些影响也被定量地解释。这些发现为SS蛋白质结构测定开辟了新的前景，包括使用分子间PCS用于具有二维或一维顺序的SS样品，如原纤维。
The number of structural restraints that can be obtained to solve 3D structures of proteins in the solid state (SS) is significantly lower than that in solution, and in relatively few cases, their skillful use has actually produced 3D structures. Paramagnetic metal ions, either as substitutes of diamagnetic metals in metalloproteins or inserted in suitably designed tags, are known to provide a wealth of structural restraints in solution, among which pseudocontact shifts are very precious. Here we show that pseudocontact shifts (pcs) generated by paramagnetic metal ions with sufficiently large spin quantum number and magnetic susceptibility anisotropy can be easily measured, and in large numbers, in SS spectra of proteins. The sample was microcrystalline cobalt(II)-substituted matrix metalloproteinase 12 (MMP-12, 17 kDa). About 250 pcs were observed for nuclei up to more than 20 A from the metal and were found in very good agreement with the calculated ones. A limited number of nuclei are also influenced by intermolecular interactions with paramagnetic metals in neighboring molecules within the crystal lattice, and these effects are also quantitatively accounted for. These findings open new perspectives for SS protein structure determinations, including the use of the intermolecular pcs for SS samples with two- or one-dimensional order, such as fibrils.