Gillespie J R, Shortle D
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Mol Biol. 1997 Apr 25;268(1):170-84. doi: 10.1006/jmbi.1997.0953.
Structural analysis of delta131delta, a fragment model of 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 T2 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(-6)-weighted, time and ensemble-averaged distance between the spin label and 30 to 60 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 in 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.
通过顺磁弛豫增强获得链段之间的长程距离限制，扩展了葡萄球菌核酸酶变性状态片段模型delta131delta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，并通过核磁共振光谱测量了酰胺质子的T2增强。当这些数据与测量的或估计的相关时间tau(c)相结合时，对于每个自旋标记的蛋白质，可以计算出自旋标记与30至60个酰胺质子之间的r(-6)加权、时间和系综平均距离。基于大约700个这样的宽松距离限制，通过距离几何/分子动力学相结合的方法生成了兼容结构的系综。由于这些距离限制的物理基础存在很大不确定性，进行了大量计算以确定计算结构对这些限制中系统误差的敏感性。总体而言，顺磁弛豫数据中反映的结构特征是稳健的；tau(c)、允许距离的边界窗口或使用的限制距离数量的大幅变化对所有计算结构共有的一般特征影响很小。与数据一致的构象系综所描述的这种变性形式的葡萄球菌核酸酶的全局拓扑结构与天然状态惊人地相似，主要区别在于两个疏水片段的分离，这两个片段在天然状态下形成一个β发夹结构。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在变性状态下就已确立。仅疏水相互作用可能编码全局拓扑结构。
