Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

James Petersson

Second Advisor

Elizabeth Rhoades

Abstract

Alpha-synuclein (αS) is an intrinsically disordered protein believed to mediate synaptic vesicle trafficking through interactions with lipid surfaces. The pathological, fibrillar aggregates of αS are implicated in a class of neurodegenerative disorders including Parkinson’s disease. A variety of post-translational modifications (PTMs) are known or hypothesized to affect both the function of αS and its propensity to form fibrils. In this work, we introduce PTMs of interest along with fluorescent probes into αS through semi-synthesis, affording site-specifically modified αS for functional and disease-relevant studies. Organic and peptide syntheses, protein expression using unnatural amino acids, and semi-synthetic strategies involving native chemical ligation (NCL) are optimized for the incorporation of challenging or novel modifications such as phosphorylation, arginylation, and acetylation. Where applicable, we evaluate trade-offs between the synthetic ease of using a mimic and the extent to which it recapitulates the properties of the authentic PTM. We characterize the aggregation of PTM-bearing αS and identify the kinetic and thermodynamic effects on fibril formation brought about by subtle changes in levels of modification. A bidirectional effect on aggregation is observed for tyrosine-39 phosphorylated αS, whereas a dose-dependent reduction in fibril formation is seen for arginylated αS, with additive effects for arginylation at more than one site. Single molecule Förster resonance energy transfer (smFRET) measurements reveal changes in monomer conformation that underlie a possible mechanism for the differential aggregation profile of phosphorylated αS. In addition to its disease-relevant characteristics, we examine the function of modified αS using fluorescence correlation spectroscopy (FCS) to assay its affinity for lipid vesicles. We find that neither phosphorylation nor arginylation alters lipid binding. However, we observe by smFRET that phosphorylation gives rise to conformational rearrangements in the presence of lipid vesicles. Finally, we elucidate the molecular basis of interactions between αS and the human acetyl transferase enzyme NatB, complemented with investigations at the single molecule level into the dynamics of a synthetic inhibitor consisting of full-length αS. Our results shed light on the substantial impact that subtle changes in PTM levels have on protein structure, native function, and fibril formation in disease.

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