Characterization of long-range structure in the denatured state of staphylococcal nuclease .2. Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures
Characterization of long-range structure in the denatured state of staphylococcal nuclease .2. Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures
复制标题
DOI：
10.1006/jmbi.1997.0953
复制
发表时间：
1997-04-25
影响因子：
5.6
通讯作者：
Shortle, D
中科院分区：
文献类型：
--
作者：
Gillespie, JR;Shortle, D
Delta 131 Delta 是一种葡萄球菌核酸酶变性状态的片段模型，通过顺磁弛豫增强获得链段之间的长程距离限制，从而扩展了 Delta 131 的结构分析。在天然状态下暴露于溶剂的位点引入了 14 个独特的 PROXYL 自旋标记，并通过 NMR 光谱测量了酰胺质子的 T-2 增强。当这些数据与测量或估计的相关时间 tau(c) 相结合时，可以计算每个自旋标记蛋白质的自旋标记与 30 至 GO 酰胺质子之间的 r(-2) 加权、时间和整体平均距离。在大约 700 个这样的松散距离约束的基础上，通过组合距离几何/分子动力学方法生成了兼容结构的集合。由于这些距离约束的物理基础存在很大的不确定性，因此进行了大量计算以确定计算结构对这些约束中的系统误差的敏感性。总体而言，顺磁弛豫数据反映的结构特征是稳健的； tau(c)、允许距离的边界窗口或所使用的约束距离数量的大变化对所有计算结构共有的一般特征影响很小。这种变性形式的葡萄球菌核酸酶的整体拓扑结构，如与构象集合所描述的一致。这些数据与天然状态的数据惊人地相似，主要区别在于在天然状态下形成β发夹的两个疏水片段的分离。这些发现表明，在没有涉及紧密堆积或稳定氢键的协同相互作用的情况下，蛋白质折叠的拓扑结构是在变性状态下建立的。仅疏水相互作用就可以编码全局拓扑。 (C) 1997 学术出版社有限公司。
Structural analysis of Delta 131 Delta, a fragment model elf the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between chain segments by paramagnetic relaxation enhancement. Fourteen unique PROXYL spin labels were introduced at sites that are solvent-exposed in the native state, and the resulting enhancements of T-2 for the amide protons were measured by NMR spectroscopy. When these data were combined with either measured or estimated correlation times tau(c), the r(-2)-weighted, time and ensemble-averaged distance between the spin label and 30 to GO amide protons could be calculated for each spin-labeled protein. On the basis of approximately 700 such loose distance restraints, ensembles of compatible structures were generated by a combined distance geometry/molecular dynamics approach. Because of the large uncertainty iri the physical basis of these distance restraints, a number of calculations were carried out to establish the sensitivity of the calculated structures to systematic errors in these restraints. Overall, the structural features reflected in the paramagnetic relaxation data were robust; large variations in tau(c), in the bounds window of allowed distances, or in the number of restraint distances used had small effects on the general features common to all calculated structures. The global topology of this denatured form of staphylococcal nuclease, as described by an ensemble of conformations consistent with. the data, is strikingly similar to that of the native state, the major difference being the segregation of two hydrophobic segments that form a beta hairpin in the native state, These findings suggest that the topology of a protein's fold is established in the denatured state in the absence of cooperative interactions involving tight packing or stable hydrogen bonding. Hydrophobic interactions alone may encode global topology. (C) 1997 Academic Press Limited.