Alpha-synuclein (αS) is an intrinsically disordered protein found at synaptic termini and thought to participate in vesicle trafficking. Changes in αS conformation and interactions with other biomolecules affect its ability to function or its propensity to form pathological aggregates, which are implicated in Parkinson’s disease. Many post-translational modifications (PTMs) have been identified in αS and are known or hypothesized to affect αS aggregate formation. We demonstrate semi-synthetic methods combining organic and peptide syntheses, unnatural amino acid mutagenesis, and native chemical ligation to introduce PTMs of interest along with fluorescent probes into αS. We characterize the aggregation kinetics of the PTM-bearing αS and identify an unusual bidirectional effect brought about by subtle changes in level of phosphorylation. Single molecule Forster resonance energy transfer (smFRET) measurements and analysis of existing fibril structures reveal the molecular interactions that underlie a possible mechanism for these effects. We also determine changes in the binding affinity of phosphorylated αS for lipid vesicles through fluorescence correlation spectroscopy and further characterize the structural changes induced by the PTM via smFRET in the presence of lipid vesicles. To understand the role of steric and electronic properties of phosphorylation, we compare our authentically phosphorylated αS to a glutamate mutant “phosphomimetic,” which is found to recapitulate some but not all of the effects of the authentic PTM. Our findings imply that subtle (mis)regulation of PTM levels in vivo have substantial impact on protein structure, dysfunction, and fibril formation in disease.

Combining semi-synthesis and single molecule biophysics allows identification of a bidirectional effect on alpha-synuclein fibril formation

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.