Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data
Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data
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DOI：
10.1021/bi000060h
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发表时间：
2000-05-09
期刊：
影响因子：
2.9
通讯作者：
Wagner, G
中科院分区：
文献类型：
--
作者：
Battiste, JL;Wagner, G
为了测试N-15异源单量子相干（HSQC）共振的顺磁展宽距离是否可以用来确定一个大的全氘代蛋白质的全局折叠，我们使用了5个氨基酸的N-15标记的真核翻译起始因子4 E（eIF 4 E）的表面上的定点自旋标记。eIF 4 E是一种25 kDa的翻译起始蛋白，其溶液结构先前在总分子量类似于45-50 kDa的3-[（3-胆酰胺丙基）二甲基铵基]-1-丙磺酸盐水合物（CHAPS）胶束中溶解。对于所有自旋标记取代，观察到与eIF 4 E的三维结构一致的距离依赖性线增宽。顺磁增宽效应（PBES）转化为距离建模通过一个简单的方法比较峰高N-15-HSQC光谱前后的氮氧自旋标记与抗坏血酸还原。PBE与HN-HN核Overhauser效应（NOE）和化学位移指数（CSI）角度限制相结合，以2.3埃的主链精度（对于二级结构元件为1.7埃）正确地确定了eIF 4 E的全局折叠。单独使用HN-HN NOE和CSI角度无法正确确定全局折叠。PBE与来自另一种核磁共振（NMR）方法的模拟约束相结合，用于大蛋白质（甲基质子化，高氘代样品）的全局折叠测定，提高了计算结构的质量。此外，从全α-螺旋蛋白质（40 kDa法呢基二磷酸合酶）的晶体结构模拟的两种方法的组合正确地确定了全局折叠，其中两种方法都不单独成功。这些结果显示了通过NMR获得40-100 kDa范围内蛋白质的中等分辨率结构的潜在可行性。
To test whether distances derived from paramagnetic broadening of N-15 heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of N-15-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass similar to 45-50 kDa. Distance-dependent Line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in N-15-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 Angstrom (1.7 Angstrom for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl-protonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all alpha-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.