Semi-synthesis is a versatile technique that allows for the site-specific incorporation of biologically relevant modifications and biophysical probes into proteins of interest. Alpha-synuclein (αS) is an intrinsically disordered protein, the pathological aggregates of which are hallmarks of Parkinson’s disease. A variety of post-translational modifications (PTMs), including phosphorylation, arginylation, nitration, and N-terminal acetylation, are believed to affect the structure of αS and its interactions with other biomolecules. We develop semi-synthetic routes combining organic synthesis, peptide synthesis, unnatural amino acid mutagenesis, and native chemical ligation to introduce PTMs of interest along with fluorescent probes into αS. Following the synthesis of unnatural amino acids bearing a PTM of interest or a bioorthogonal handle, we incorporate them into αS via peptide synthesis or amber codon suppression. We also present a novel chemoenzymatic strategy to synthesize αS bearing one PTM and two fluorophores with improved yield. To compare αS bearing authentic PTMs to their corresponding mimics, we make such mimics both through common mutations and by synthesizing a small molecule that we react with recombinantly produced protein. Through biophysical measurements, we characterize the conformation, aggregation kinetics, and vesicle binding affinities of our multiply modified αS to elucidate how subtle differences in PTM levels dramatically alter the behavior of the protein. Our study demonstrates the robust reliability of incorporating authentic PTMs via synthetic strategies and provides an assessment of the utility of certain chemical modification mimics in studying biological processes.

Semi-synthesis of post-translationally modified and fluorescently labeled alpha-synuclein enables identification of protein behavior regulation by subtle changes in modification levels

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.