Biophysical Determinants of Physiological and Pathological alpha-Synuclein Membrane Interactions

α-突触核蛋白是一种丰富的神经元蛋白，与神经退行性疾病有关，具有多种构象状态，参与其生理和病理活动。我们最近使用深度突变扫描在细胞模型中探索了驱动其毒性的α-突触核蛋白的构象，令人惊讶的是，我们发现这种异常的表型是由一个动态的膜结合的两亲性螺旋驱动的，这也被认为是介导其在囊泡运输中的天然生理作用。α-突触核蛋白的生理构象如何参与病理？我们的数据突出了α-突触核蛋白的一个关键序列特征，我们假设它通过增加膜结合螺旋的动力学来调节其与脂膜的生理和病理相互作用。在这个提案中，我们将检验我们的假设，即螺旋动力学的分子基础，以及这些动力学对胞吐和聚集的贡献。为了验证这些假设，首席研究员(PI)需要在核磁共振波谱、哺乳动物细胞培养和操作以及细胞成像方面进行额外的培训，以及在膜蛋白生物物理学和囊泡运输和神经传递的分子生物学方面的额外专业知识。因此，这些假说将在世界领先的膜蛋白结构和功能专家威廉·德格拉多教授和世界领先的α-突触核蛋白在胞吐中的作用专家罗伯特·爱德华兹教授的指导下得到解决。在他们的指导下，PI将(1)利用核磁共振波谱测试α-突触核蛋白序列特征对膜结合状态动力学的贡献，以及(2)测试这些序列特征在介导α-突触核蛋白对神经内分泌细胞胞吐作用中的作用。指导阶段完成后，PI将(3)使用深度突变扫描测试动态膜结合对α-突触核蛋白聚集的贡献。总之，这些目标将提供一种分子机制，通过这种机制，α-突触核蛋白的独特结构有助于其生理和病理活动。此外，这些经验提供的培训将使PI能够开始一项独立的科学生涯，研究蛋白质和脂膜之间的功能相互作用，以及蛋白质错误折叠的生物物理和细胞决定因素。

The abundant neuronal protein α-synuclein, which is implicated in neurodegenerative disease, adopts a wide variety of conformational states that contribute to its physiological and pathological activities. We recently used deep mutational scanning to probe the conformation of α-synuclein that drives its toxicity in a cellular model, and surprisingly, we found that this aberrant phenotype is driven by a dynamic, membrane-bound amphiphilic helix, which is also believed to mediate its native physiological role in vesicle trafficking. How does the physiological conformation of α-synuclein contribute to pathology? Our data highlight a critical sequence feature of α-synuclein that we hypothesize mediates both its physiological and pathological interactions with lipid membranes by increasing dynamics of the membrane-bound helix. In this proposal, we will test our hypothesis for the molecular basis of helix dynamics, as well as the contribution of those dynamics to exocytosis and aggregation. In order to test these hypotheses, the principal investigator (PI) requires additional training in nuclear magnetic resonance (NMR) spectroscopy, mammalian cell culture and manipulation, and cellular imaging, as well as additional expertise in membrane protein biophysics and the molecular biology of vesicle trafficking and neurotransmission. These hypotheses will therefore be addressed under the mentorship of Prof. William DeGrado, one of the world’s leading experts in membrane protein structure and function, and Prof. Robert Edwards, one of the world’s leading experts on the role of α-synuclein in exocytosis. With their guidance, the PI will (1) test the contribution of α-synuclein sequence features to the dynamics of the membrane bound state using NMR spectroscopy, and (2) test the role of those sequence features in mediating α-synuclein’s effect on exocytosis in neuroendocrine cells. Following completion of the mentored phase, the PI will (3) test the contributions of dynamic membrane binding to α-synuclein aggregation using deep mutational scanning. Together, these aims will provide a molecular mechanism by which the unique structure of α-synuclein contributes to both its physiological and pathological activities. Moreover, the training provided by these experiences will position the PI to launch an independent scientific career examining functional interactions between proteins and lipid membranes, as well as the biophysical and cellular determinants of protein misfolding.