Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule and Computational Approaches for a Disordered Protein Region.
Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule and Computational Approaches for a Disordered Protein Region.
复制标题
DOI：
10.1021/jacs.5b04471
复制
发表时间：
2015-09-23
影响因子：
15
通讯作者：
Rhoades E
中科院分区：
文献类型：
--
作者：
Metskas LA;Rhoades E
近年来，单分子Förster共振能量转移(SmFRET)已经成为结构生物学中的一种重要而灵活的工具，特别是在研究高动态区域和分子组装方面。通过将smFRET与计算方法相结合，将粗粒度的实验数据与更高分辨率的SOLICO计算相结合，可以进一步扩大smFRET的用途。在这里，我们使用smFRET来确定心肌肌钙蛋白复合体的肌钙蛋白I亚单位(TnIC)固有无序的C-末端结构域内的六个成对距离。我们使用已发表的TnIC的相互冲突的结构作为分子动力学模拟的起始模型，在提取构象动力学信息之前，通过与smFRET测量的成功比较来验证该模型的有效性。我们发现，在Silico中，残基之间的成对距离波动很大，但在跨时间平均后，模拟结果与更长时间尺度的smFRET测量结果总体上是一致的。最后，蒙特卡罗模拟证明，TnIC的低能构象确实是不同的，但最高采样的星团类似于已公布的相互冲突的模型。通过这种方式，我们发现有争议的结构只是这个动态区域的局部极小值，并且包括所有这三个结构的群体仍然与光谱测量一致。综上所述，这里描述的组合方法使我们能够批判性地评估TnIC的现有模型，从而洞察TnIC在其可能的无序-有序转变之前的无序态的构象和动力学。此外，它们还提供了一个框架，将计算和实验方法与不同的时间尺度相结合，用于研究无序和动态的蛋白质状态。
In recent years, single-molecule Förster resonance energy transfer (smFRET) has emerged as a critical and flexible tool in structural biology, particularly in the study of highly dynamic regions and molecular assemblies. The usefulness of smFRET can be further extended by combining it with computational approaches, marrying the coarse-grained experimental data with higher-resolution in silico calculations. Here we use smFRET to determine six pairwise distances within the intrinsically disordered C-terminal domain of the troponin I subunit (TnIC) of the cardiac troponin complex. We used published conflicting structures of TnIC as starting models for molecular dynamics simulations, which were validated through successful comparison with smFRET measurements before extracting information on conformational dynamics. We flnd that pairwise distances between residues fluctuate widely in silico, but simulations are generally in good agreement with longer time scale smFRET measurements after averaging across time. Finally, Monte Carlo simulations establish that the lower-energy conformers of TnIC are indeed varied, but that the highest-sampled clusters resemble the published, conflicting models. In this way, we flnd that the controversial structures are simply stabilized local minima of this dynamic region, and a population including all three would still be consistent with spectroscopic measurements. Taken together, the combined approaches described here allow us to critically evaluate existing models of TnIC, giving insight into the conformation and dynamics of TnIC’s disordered state prior to its probable disorder–order transition. Moreover, they provide a framework for combining computational and experimental methods with diflerent time scales for the study of disordered and dynamic protein states.