Structural Biology and Biophysics of Alpha-Synuclein Fibrils by Solid-State NMR

摘要 联体核病是一个主要的公共健康风险，每年有数百万人受到帕金森病的影响， 帕金森痴呆症(PDD)、路易体痴呆和多系统萎缩。这些疾病 影响运动和认知功能，目前还没有已知的治疗方法和有限的治疗选择。它 因此，对于确定疾病病因至关重要，这被认为是由于错误折叠和 蛋白质α-突触核蛋白聚集成纤维。这些致病原纤维的体内结构形式将有助于 为了了解错误折叠的机制并帮助开发具有更高结构的成像配体- 特定的绑定。我将使用固体核磁共振(SS核磁共振)结合冷冻-EM/ET来确定结构 患者体内获得的PDD原纤维的比例。然后我将研究体内纤维四级结构是如何支配 成熟病态纤维的稳定性、动力学及其对α-突触核蛋白聚集途径的影响 纤维。我还将研究成像配体与这些纤维的相互作用，以确定结构基序 这些化合物通过比较体外和体内纤维制剂的结合部位结构来结合。 培训计划：我在膜蛋白的SS核磁共振方面有相当多的研究经验，我 将增加使用新技术确定纤维结构所需的SS核磁共振方法的培训 将模拟退火法和分子动力学与低温电子显微镜(Cryo-EM)相结合的方法， 低温电子断层扫描(CRYO-ET)和SS核磁共振数据。位于麦迪逊的国家磁共振设施 (NMRFAM)提供世界领先的培训环境，可使用高场SSNMR谱仪 (600至900 MHz)和一台超高场(1.1 GHz)SS核磁共振光谱仪，将于2023年交付。基础设施 这将使我能够对患病的纤维和它们的结构和动力学做出新的发现 与成像配体的相互作用。此外，我将接受低温培训，以获得补充数据，如 如纤丝宽度、扭度、单位长度质量，并利用Cryo-EM与SS核磁共振联合对原子进行结构求解 决议。这些发现将通过出版物和讲座向更大的科学界传播。 在跨学科的会议上。我的培训将在国际公认的查德·里恩斯特拉教授的指导下进行 生物分子核磁共振领域的领军人物，曾指导过数十名研究生和博士后，其中许多 其中一些人是顶尖学府的教职员工。环境：威斯康星大学麦迪逊分校是 排名很高的研究型大学，拥有世界上最好的结构生物学环境， 与NMRFAM、低温电磁研究中心和高吞吐量计算中心合作。凯瑟琳教授 Henzler-Wildman和Rienstra教授是NMRFAM的联合主任，他在不溶性蛋白质方面有很强的背景 结构和动力学。与蒂莫西·格兰特教授的低温EM小组就纤维结构进行密切合作 决心将是一种额外的能力。威斯康星大学麦迪逊分校良好的研究环境。 Rienstra将使我做好准备，成为未来结构生物学和复杂生物分子生物物理学的领导者。

ABSTRACT Synucleinopathies are a major public health risk, with millions of people each year affected by Parkinson disease, Parkinson disease with dementia (PDD), Lewy Body dementia, and multiple system atrophy. These diseases impact both motor and cognitive function, for which there are no known cures and limited therapeutic options. It is therefore vital to determine the disease etiology, which is hypothesized to arise from the misfolding and aggregation of the protein α-synuclein into fibrils. The in vivo structural forms of these pathogenic fibrils will help to understand mechanisms of misfolding and aid in the development of imaging ligands with higher structure- specific binding. I will use solid state NMR (SSNMR) in combination with cryo-EM/ET to determine the structures of patient derived in vivo PDD fibrils. I will then investigate how in vivo fibril quaternary structure governs the stability and dynamics of mature diseased state fibrils and its effects on the aggregation pathway of α-synuclein fibrils. I will also investigate the interactions of imaging ligands to these fibrils to determine the structural motifs these compounds bind to by comparing binding site structure between in vitro and in vivo fibril preparations. Training plan: I have a considerable amount of research experience with SSNMR of membrane proteins, and I will add training in SSNMR methods required for structure determination of fibrils structures using novel approaches combining simulated annealing and molecular dynamics with cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET) and SSNMR data. The National Magnetic Resonance Facility at Madison (NMRFAM) provides a world-leading environment for training with access to high field SSNMR spectrometers (600 to 900 MHz) and an ultra-high field (1.1 GHz) SSNMR spectrometer arriving in 2023. The infrastructure here will allow me to make new discoveries to both structure and dynamics of diseased fibrils and their interactions with imaging ligands. Furthermore, I will gain training in cryo-ET to obtain complementary data such as fibril width, twist, mass-per-unit length, and utilize cryo-EM to solve structures jointly with SSNMR to atomic resolution. These findings will be disseminated to the larger scientific community via publications and talks given at interdisciplinary meetings. My training will take place under Prof. Chad Rienstra, an internationally recognized leader in the field of biomolecular SSNMR, who has mentored dozens of graduate students and postdocs, many of whom are faculty members at top tier institutions. Environment: The University of Wisconsin-Madison is a highly ranked research university, with among the best environments available in the world for structural biology, with NMRFAM, the Cryo-EM Research Center, and the Center for High Throughput Computing. Prof. Katherine Henzler-Wildman, co-director of NMRFAM with Prof. Rienstra, has a strong background in insoluble protein structure and dynamics. Close collaboration with the cryo-EM group of Prof. Timothy Grant on fibril structure determination will be an added capability. The excellent research environment at UW-Madison with Prof. Rienstra will prepare me to be a future leader in structural biology and biophysics of complex biomolecules.