Backbone-only protein solution structures with a combination of classical and paramagnetism-based constraints: A method that can be scaled to large molecules
Backbone-only protein solution structures with a combination of classical and paramagnetism-based constraints: A method that can be scaled to large molecules
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DOI：
10.1002/cphc.200301058
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发表时间：
2004-06-21
期刊：
影响因子：
2.9
通讯作者：
Parigi, G
中科院分区：
文献类型：
--
作者：
Barbieri, R;Luchinat, C;Parigi, G
本文表明，如果具有牢固结合的顺磁金属的衍生物可用，则可以通过单独分配蛋白质主链和主链相关约束来获得蛋白质的中等分辨率解决方案结构。钙结合蛋白 D-9k 提供了概念验证，钙结合蛋白 D-9k 是一种钙结合蛋白，其中两个钙离子之一可以选择性地被顺磁性镧系元素离子取代。使用的约束是 H-N（和 H-alpha）核。欧沃豪瑟效应 (NOE)、氢键、化学位移的二面角约束以及以下基于顺磁性的约束：1) 赝接触位移，通过在 C 末端钙结合位点取代一个（或多个）镧系元素而获得； 2) 由于顺磁性镧系元素引起的自取向而产生 N-H-N 残余偶极耦合； 3）居里和核间偶极-偶极相互作用之间的互相关； 4）顺磁性引起的弛豫率增强。还根据蛋白质的分子量给出了任意两个主链原子之间的核间距离的上限。为此，基于顺磁性的约束在 CYANA 程序中共同实施，用于确定解决方案结构，类似于之前为 DYANA 程序所做的操作。该方法本质上适用于大分子量蛋白质。
Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constraints if a derivative with a firmly bound paramagnetic metal is available. The proof-of-concept is provided on calbindin D-9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are H-N (and H-alpha) nuclear. Overhauser effects (NOEs), hydrogen bonds, dihedral angle constraints from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) N-H-N residual dipolar couplings due to self-orientation induced by the paramagnetic lanthanides(s); 3) cross-correlations between the Curie and internuclear dipole-dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins.