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

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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.