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

摘要 突触核蛋白病是一种主要的公共卫生风险，每年有数百万人受到帕金森病的影响， 帕金森病伴痴呆（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，对此没有已知的治愈方法，治疗选择有限。它 因此，确定疾病病因是至关重要的，这是假设产生的错误折叠， 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 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- 特异性结合。我将使用固态核磁共振（SSNMR）结合冷冻EM/ET来确定结构 患者体内来源的PDD原纤维。然后，我将研究在体内原纤维四级结构如何支配 成熟病态纤维的稳定性和动力学及其对α-突触核蛋白聚集途径的影响 纤维我也将研究这些纤维的成像配体的相互作用，以确定结构基序 这些化合物通过比较体外和体内原纤维制备物之间的结合位点结构而结合。 培训计划：我有相当多的膜蛋白SSNMR研究经验，我 将增加SSNMR方法的培训，这些方法是使用新方法测定原纤维结构所需的 将模拟退火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SSNMR数据。位于麦迪逊的国家磁共振设备 （NMRFAM）提供了一个世界领先的培训环境，可以使用高场SSNMR光谱仪 (600到900 MHz）和超高场（1.1 GHz）SSNMR光谱仪将于2023年抵达。基础设施 这将使我对患病纤维的结构和动力学及其 与成像配体的相互作用。此外，我将获得冷冻ET培训，以获得补充数据， 如纤维宽度，扭曲，每单位长度的质量，并利用冷冻EM解决结构与SSNMR联合原子 分辨率这些发现将通过出版物和演讲传播给更大的科学界。 在跨学科的会议上。我的培训将在Chad Rienstra教授的指导下进行，他是一位国际公认的 生物分子SSNMR领域的领导者，他指导了数十名研究生和博士后，其中许多人 他们中的许多人都是顶级院校的教员。环境：威斯康星大学麦迪逊分校是一所 排名靠前的研究型大学，拥有世界上结构生物学最好的环境之一， 与NMRFAM、Cryo-EM研究中心和高吞吐量计算中心合作。凯瑟琳教授 Henzler-Wildman与Rienstra教授共同担任NMRFAM的主任，他在不溶性蛋白质方面有很强的背景 结构和动力学。与Timothy Grant教授的cryo-EM小组在原纤维结构方面密切合作 决心将是一种额外的能力。在威斯康星大学麦迪逊分校与教授优秀的研究环境。 Rienstra将使我成为未来复杂生物分子的结构生物学和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.