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

通过固态核磁共振研究 α-突触核蛋白原纤维的结构生物学和生物物理学

• 批准号: 10605819
• 负责人: Collin Griffin Borcik
• 金额: $ 6.91万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605819
• 关键词: Affect; Affinity; Amyloid; Binding; Binding Sites; Biophysics; Chad; Characteristics; Chemicals; Collaborations; Communities; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data; Development; Diagnosis; Diagnostic; Disease; Environment; Etiology; Exhibits; Faculty; Fellowship; Fluorescent Dyes; Formulation; Future; Grant; Imaging ligands; In Vitro; Infrastructure; Institution; International; Joints; Label; Lansoprazole; Length; Lewy Body Dementia; Ligand Binding; Ligands; Location; Magnetic Resonance; Measures; Membrane Proteins; Mentors; Methodology; Methods; Molecular Conformation; Motor; Multiple System Atrophy; NMR Spectroscopy; National Research Service Awards; Neurodegenerative Disorders; Parkinson Disease; Parkinson' s Dementia; Pathogenicity; Pathology; Pathway interactions; Patients; Persons; Phenotype; Physiological; Pittsburgh Compound-B; Polymorph; Positron-Emission Tomography; Postdoctoral Fellow; Preparation; Property; Protein Dynamics; Public Health; Publications; Relaxation; Research; Residual state; Resolution; Risk; Site; Spectrum Analysis; Structure; Techniques; Technology; Temperature; Testing; Therapeutic; Tissues; Training; Universities; Variant; Vertebral column; Water; Width; Wisconsin; aggregation pathway; alpha synuclein; clinical application; cognitive function; diagnostic tool; disease phenotype; experience; experimental study; graduate student; improved; in vivo; insight; meetings; member; molecular dynamics; novel; novel strategies; preference; protein aggregation; protein structure; small molecule; solid state nuclear magnetic resonance; structural biology; synucleinopathy; tool; tool development

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.

抽象的 突触核心病是一种主要的公共卫生风险，每年数百万的人受帕金森病的影响， 患有痴呆症（PDD），路易体内痴呆和多种系统萎缩的帕金森病。这些疾病 影响运动功能和认知功能，对于这些功能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，疾病的病因是由错误折叠和 将蛋白α-突触核蛋白聚集到原纤维中。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和有助于发展具有较高结构的成像配体的发展 - 特定的结合。我将使用固态NMR（SSNMR）与Cryo-EM/ET结合使用来确定结构 在体内PDD纤维中得出的患者。然后，我将研究体内原纤维季季结构如何控制 稳定性和成熟状态原纤维的动力学及其对α-突触核蛋白聚集途径的影响 原纤维。我还将研究成像配体与这些原纤维的相互作用，以确定结构基序 这些化合物通过比较体外和体内原纤维制剂之间的结合位点结构来结合。 培训计划：我对膜蛋白的SSNMR有大量的研究经验，我 将在结构确定原纤维结构所需的SSNMR方法中添加训练 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， 冷冻电子层析成像（Cryo-ET）和SSNMR数据。麦迪逊的国家磁共振设施 （NMRFAM）提供了一个世界领先的环境，用于访问高场SSNMR光谱仪的训练环境 （600至900 MHz）和2023年到达的超高场（1.1 GHz）SSNMR光谱仪。基础设施 这将使我能够对患病原纤维的结构和动态进行新发现 与成像配体的相互作用。此外，我将在Cryo-Et中获得培训，以获取完整的数据此类数据 作为原纤维宽度，扭曲，质量单位的长度，并利用冷冻EM与SSNMR共同求解结构 解决。这些发现将通过出版物和谈判传播给更大的科学界 在跨学科会议上。我的培训将在国际公认 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 其中之一是顶级机构的教职员工。环境：威斯康星大学麦迪逊分校是 排名较高的研究大学，拥有世界上最好的结构生物学环境之一 使用NMRFAM，冷冻EM研究中心和高通量计算中心。凯瑟琳教授 NMRFAM与Rienstra教授的联合主任Henzler-Wildman在不溶性蛋白方面具有强大的背景 结构和动态。与蒂莫西·格兰特（Timothy Grant）教授的冷冻EM集团密切合作 确定将是一个增加的功能。 UW-Madison的出色研究环境与教授 rienstra将使我成为复杂生物分子的结构生物学和生物物理学的未来领导者。