Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α‑Synuclein

In Parkinson’s disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.在帕金森病及其他突触核蛋白病（synucleinopathies）中，α-突触核蛋白（α-synuclein）会发生错误折叠并聚集。然而，其固有的无序特性使其能够采取多种由内部氢键稳定的亚稳态构象。由于这些构象在短时间尺度内相互转换，无序蛋白的单体构象难以通过常规结构技术进行表征。能够检测单体α-突触核蛋白构象的技术寥寥无几，其中包括毫秒级氢氘交换质谱（millisecond hydrogen/deuterium-exchange mass spectrometry, HDX-MS）。本研究中，我们开发了一种将毫秒级氢氘交换质谱数据与聚集动力学相关联的新方法，用于解析全长野生型（wild-type）单体α-突触核蛋白自组装过程背后的局部结构动态。我们定制的仪器与软件能够在生理条件（模拟细胞外、细胞内及溶酶体（lysosomal）细胞区室的环境）下，以残基分辨率（residue resolution）测定野生型α-突触核蛋白单体的毫秒级酰胺氢交换速率。我们采用经验校正方法对测得的氢交换速率进行归一化处理，从而能够在差异极大的溶液条件间进行比较。我们对所得淀粉样原纤维的聚集动力学与形态进行了表征，并将其与单体的结构变化相关联。本研究首次采用关联方法，将α-突触核蛋白的分子构象与其聚集过程相关联，我们发现α-突触核蛋白的中央C端残基是其成核及聚集过程的核心驱动因素。我们提出了一种将固有无序蛋白的局部结构动态与其功能特性相关联的新方法，并通过单体α-突触核蛋白局部化学环境与构象系综偏倚（conformational ensemble bias）之间关系的全新细节，为该方法提供了实验佐证。