The maintenance of proteins in their stable, folded state is essential to proper cellular function. Protein misfolding underlies at least 11 neurodegenerative disorders, including Lewy Body dementia and Parkinson's disease. Current Parkinson's disease treatments provide only symptom relief, and significant side effects are observed. Drugs that reverse or block aggregation, combined with early diagnosis, provide the best prospect for a cure that preserves the patient's memories. To design such drugs, one must understand the process of aggregation and propagation. We propose to use novel fluorescence techniques developed in our laboratory to structurally characterize aggregates of the protein ?-synuclein (?S), the pathological hallmark of Parkinson's disease. ?S monomers misfold, forming small aggregates of a few monomer units (oligomers) before going on to form long fibrils that can create insoluble tangles that are toxic to neurons. Determining the molecular structure of these oligomers is crucial for elucidating the aggregation pathway involved in fibril formation. Furthermore, structural characterization of the membrane-crossing behavior of ?S may help to explain the spread of pathology from neuron-to-neuron. However, experimental and theoretical efforts are complicated by stoichiometric heterogeneity, the presence of unstructured regions, and the temporal instability of oligomers. Our recent development of thioamide fluorescence quenching as a time-dependent structure determination method offers a powerful new tool to be applied to amyloid characterization. Since thioamides can be inserted at any position in the peptide backbone, they can provide minimally-perturbing, single residue probes of intra- and intermolecular contacts. Fluorescence techniques are well suited to address three of the outstanding problems with oligomer structure determination: heterogeneity (because single-molecule techniques can be used), sample environment (because studies can be carried out under dilute conditions in cell cultures), and temporal instability (changes can be monitored in real time). Using thioamide fluorescence quenching, we will generate structural models of the aggregation intermediates and validate these models with fluorescence measurements made in cultured cells.
The aggregation of amyloid peptides into oligomers (clusters) and ultimately into fibrillar tangles called Lewy bodies plays a pivotal role in the development of Parkinson's disease. The proposed research will study the structures of intermediates in the aggregation process in vitro and in cultured cells to aid in the design of therapies targeted toward alleviating their toxicity.
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