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This dissertation work aims to advance the current understanding of the native function of α-Synuclein (αS), an intrinsically disordered protein whose intraneuronal aggregation is most notably recognized as a pathological hallmark of Parkinson’s disease, among many other neurodegenerative disorders classified as synucleopathies. The putative function of αS is its interaction with synaptic vesicles, though it remains unclear on the molecular level how its membrane binding and surface activity can specifically regulate synaptic vesicle organization and homeostatic mechanisms at large. From a membrane biophysics perspective, the partial folding of αS leading to helix formation on synaptic membrane surface presents several interesting questions in the context of functional relevance of its membrane bound structure. While the first ~100 residues participate in lipid binding, the remaining 40 residues (the C-terminal domain) retain the protein’s intrinsic disorder, creating a physisorbed polymer on the membrane surface. How the sterically projecting C-terminal domain can mediate the interactions of synaptic vesicles with one another is an important fundamental question we address. We probe αS interactions with model membranes mimicking synaptic vesicles using biophysical approaches inspired by nano-bio interface, X-ray scattering, and polymer physics. We first produce and validate a silica nanoparticle-based model membrane system that mimics the curvature and composition of a synaptic vesicle using electron microscopy and isothermal titration calorimetry. We then examine the surface activity of membrane bound αS by a combination of small angle X-ray scattering, depletion force response, and X-ray photon correlation spectroscopy, in order to characterize the polymeric nature of the C-terminal domain. We find that our model system, spherical supported lipid bilayers (SSLBs), can be efficiently prepared by a generalizable osmotic stress approach. We demonstrate that a critical density of αS on SSLB surface confers complete steric stabilization of SSLBs, which is driven by the polymeric properties of the C-terminal domain that were assessed from quantitating its steric effect with depletion force measurements. Overall, our findings implicate the role of αS in the release of synaptic vesicles from clustered pools within the presynaptic terminal — an important physiological step in the propagation of neurotransmission. The biophysical insights obtained from fundamental αS-membrane interaction experiments establish structure-property relationships in the context of the synaptic vesicle organization.




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