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Phosphorylation, acetylation, and other PTMs regulate \u03b1S\u2019s interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate \u03b1S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence \u03b1S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in \u03b1S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.<\/jats:p>","DOI":"10.64898\/2026.06.24.734353","type":"posted-content","created":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T15:30:39Z","timestamp":1782401439000},"source":"Crossref","is-referenced-by-count":0,"title":["Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified \u03b1-Synuclein"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9156-0895","authenticated-orcid":false,"given":"Bernard","family":"Abakah","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8471-9375","authenticated-orcid":false,"given":"Marie","family":"Shimogawa","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0769-5023","authenticated-orcid":false,"given":"Paola","family":"Miranda-Castrodad","sequence":"additional","affiliation":[{"name":"Graduate Group in Biochemistry, Biophysics, and Chemical Biology, Perelman School of Medicine, University of Pennsylvania, 206 Anatomy-Chemistry Building, 3620 Hamilton Walk, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1741-0650","authenticated-orcid":false,"given":"Elizabeth","family":"Rhoades","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA"},{"name":"Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3854-9210","authenticated-orcid":false,"given":"E. 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