Pulsed Hydrogen-Deuterium Exchange Illuminates the Aggregation Kinetics of α-Synuclein, the Causative Agent for Parkinson's Disease.
Pulsed Hydrogen-Deuterium Exchange Illuminates the Aggregation Kinetics of α-Synuclein, the Causative Agent for Parkinson's Disease.
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DOI：
10.1021/acschemneuro.8b00052
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发表时间：
2018-06-20
影响因子：
5
通讯作者：
Gross ML
中科院分区：
文献类型：
--
作者：
Illes-Toth E;Rempel DL;Gross ML
α-突触核蛋白（aS）形成毒性中间体，范围从小的寡聚体和原纤维到大的淀粉样蛋白原纤维。了解aS原纤维形成的时间过程及其区域所起的作用对于治疗干预至关重要。在这里，我们首次使用脉冲氢-氘交换和质谱（HDX-MS）来探测体外完全aS聚集的动力学中间体，实现包含关键阶段的空间分辨蛋白质信息的动力学快照。监测由此产生的质量变化显示出不同的二项式丰度两个主要的交换配置文件，一个是代表一个快速交换，溶剂访问的物种和另一个更受保护的性质。我们表明，使用一系列的蛋白水解肽的完整蛋白质，自协会是最明显的NAC区域，因此，无论是终端。然而，N-末端在中期和晚期显示出较小的保护群体，而C-末端主要显示单峰HDX，表明这些区域没有任何大的构象重排。聚焦于疏水核，我们确认并模拟了不同的同位素分布，并计算了它们的相对分数，以辨别它们各自的贡献。数据拟合报告了各自的t1/2值，这些值几乎相同且不依赖于位置。我们通过互补透射电子显微镜观察聚集体的形态，并通过圆二色性来评估二级结构的变化。我们的研究结果提供了体外aS聚集的详细情况，并证明HDX-MS在溶液中提供了独特的空间分辨，共存的动力学中间体。这个新平台适用于测试有前途的aS聚集抑制剂。
Alpha-synuclein (aS) forms toxic intermediates ranging from small oligomers and protofibrils to large amyloid fibrils. Understanding the time course of aS fibril formation and the role played by its regions is critical for therapeutic intervention. Here, we used pulsed hydrogen-deuterium-exchange and mass spectrometry (HDX-MS) for the first time to probe kinetic intermediates of the full aS aggregation in vitro, achieving kinetic snapshots containing spatially resolved protein information about critical stages. Monitoring the resultant mass shifts show distinct binomial abundances for two main exchange profiles, one that represents a fast-exchanging, solvent-accessible species and another with a more protected nature. We show using a series of proteolytic peptides from the full protein that self-association is most pronounced in the NAC region and less so for either termini. The N-terminus, however, shows a minor protected population at mid- and late times, whereas the C-terminus shows predominantly unimodal HDX, indicating that these regions are devoid of any large conformational rearrangements. Focusing on the hydrophobic core, we confirmed and modeled the different isotopic distributions and calculated their relative fractions to discern their individual contributions. The data fitting reports respective t1/2 values, which are nearly identical and do not depend on location. We followed the aggregation by complementary transmission electron microscopy to observe the morphology of aggregates and circular dichroism to assess changes in secondary structure. Our results provide a detailed picture of aS aggregation in vitro, and demonstrate that HDX-MS offers unique spatially resolved, co-existing kinetic intermediates in solution. This new platform is suitable for testing promising inhibitors of aS aggregation.