Marsh Joseph A, Neale Chris, Jack Fernando E, Choy Wing-Yiu, Lee Anna Y, Crowhurst Karin A, Forman-Kay Julie D
Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
J Mol Biol. 2007 Apr 13;367(5):1494-510. doi: 10.1016/j.jmb.2007.01.038. Epub 2007 Jan 20.
Due to their dynamic ensemble nature and a deficiency of experimental restraints, disordered states of proteins are difficult to characterize structurally. Here, we have expanded upon our previous work on the unfolded state of the Drosophila drk N-terminal (drkN) SH3 domain with our program ENSEMBLE, which assigns population weights to pregenerated conformers in order to calculate ensembles of structures whose properties are collectively consistent with experimental measurements. The experimental restraint set has been enlarged with newly measured paramagnetic relaxation enhancements from Cu(2+) bound to an amino terminal Cu(2+)-Ni(2+) binding (ATCUN) motif as well as nuclear Overhauser effect (NOE) and hydrogen exchange data from recent studies. In addition, two new pseudo-energy minimization algorithms have been implemented that have dramatically improved the speed of ENSEMBLE population weight assignment. Finally, we have greatly improved our conformational sampling by utilizing a variety of techniques to generate both random structures and structures that are biased to contain elements of native-like or non-native structure. Although it is not possible to uniquely define a representative structural ensemble, we have been able to assess various properties of the drkN SH3 domain unfolded state by performing ENSEMBLE minimizations of different conformer pools. Specifically, we have found that the experimental restraint set enforces a compact structural distribution that is not consistent with an overall native-like topology but shows preference for local non-native structure in the regions corresponding to the diverging turn and the beta5 strand of the folded state and for local native-like structure in the region corresponding to the beta6 and beta7 strands. We suggest that this approach could be generally useful for the structural characterization of disordered states.
由于蛋白质的无序状态具有动态集合性质且缺乏实验约束，因此难以对其进行结构表征。在此，我们利用我们的ENSEMBLE程序扩展了之前关于果蝇drk N端（drkN）SH3结构域未折叠状态的研究工作，该程序为预先生成的构象赋予群体权重，以便计算其性质与实验测量结果总体一致的结构集合。实验约束集已通过新测量的与氨基末端Cu(2+)-Ni(2+)结合（ATCUN）基序结合的Cu(2+)的顺磁弛豫增强以及近期研究中的核Overhauser效应（NOE）和氢交换数据得到扩充。此外，还实施了两种新的伪能量最小化算法，这显著提高了ENSEMBLE群体权重分配的速度。最后，我们通过利用多种技术生成随机结构以及偏向包含类似天然或非天然结构元素的结构，极大地改进了构象采样。尽管不可能唯一地定义一个代表性的结构集合，但我们能够通过对不同构象池进行ENSEMBLE最小化来评估drkN SH3结构域未折叠状态的各种性质。具体而言，我们发现实验约束集强制形成一种紧凑的结构分布，该分布与整体类似天然的拓扑结构不一致，但在对应于折叠状态的发散转角和β5链的区域表现出对局部非天然结构的偏好，而在对应于β6和β7链的区域表现出对局部类似天然结构的偏好。我们认为这种方法可能对无序状态的结构表征普遍有用。
