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Abstract
This dissertation work aims to explore the interactions of α-synuclein, an intrinsically disordered protein found primarily in neurons, with model lipid vesicles. α-synuclein is of particular interest to researchers due to its implicated role Parkinson’s disease, dementia with Lewy bodies, and other common neurodegenerative disorders classified as synucleinopathies. Current knowledge suggests that α-synuclein plays a role in the synaptic vesicle cycle by binding to loaded synaptic vesicles ready for exocytosis, but more work is required to elucidate the protein’s exact function in vivo. One particularly interesting hypothesis that has emerged in recent years is that α-synuclein may be able to differentiate between potential vesicular binding partners by sensing their size or curvature; ample experimental evidence supports this phenomenon, but the exact mechanism that α-synuclein uses to “sense” such small changes in curvature remains unknown.
In this work, I will describe several lines of evidence to support the hypothesis that α-synuclein senses membrane curvature by probing differences in membrane hydrophobic defect presentation. First, I will describe a novel confocal binding assay developed to explicitly study α-synuclein binding on a per-vesicle level; this method offers many advantages over more traditional protein binding assays, including very low sample consumption as well as the ability to quantify the total amount of protein bound under membrane-saturating conditions. When this new methodology is used in conjunction with molecular dynamics simulations to quantify membrane defect presentation, we find that membranes displaying more and larger defects display more bound protein at saturation than membranes of similar composition displaying fewer defects. Additionally, we explore the impact of a hypo-osmotic pressure and subsequent vesicle swelling on membrane defect presentation and α-synuclein binding. When similar experiments using the confocal binding assay are performed on hypo-osmotically stressed vesicles, we find that the resulting differences in membrane defect presentation are likely too small to meaningfully impact α-synuclein binding on a macroscopic scale. Overall, these findings support the idea that α-synuclein senses curvature through membrane defect presentation, and suggests that both the lipid composition and osmotic pressure of synaptic vesicles may play a role in helping α-synuclein to determine its physiological binding partners.