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Whereas most conventional structural biology approaches offer limited resolution in living systems, deep mutational scanning can offer insight into the folding state of a protein in living cells, and we apply this method to probe concentration\u2010dependent changes in the folding of \u03b1\u2010synuclein in a popular yeast model of pathology. We discover that at a wide range of cellular concentrations, \u03b1\u2010synuclein is highly biased toward formation of a membrane\u2010bound amphiphilic helix that imparts toxicity. Population of this toxic state can be disrupted by mutations that reduce membrane affinity, which shift the folding equilibrium away from the membrane\u2010bound state. Reduced\u2010affinity variants exhibit distinct sensitivity to concentration relative to variants with WT\u2010like affinity, likely because these variants are expressed at concentrations closer to their dissociation constant for membrane binding. These results show how mutational scanning can provide high\u2010resolution insights into the folding landscape of proteins in living cells, which is likely to be of special utility for studying proteins that misfold and\/or aggregate.<\/jats:p>","DOI":"10.1002\/pro.70456","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T13:24:23Z","timestamp":1768915463000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Concentration\u2010dependent mutational scanning probes the cellular folding landscape of \u03b1\u2010synuclein in yeast"],"prefix":"10.1002","volume":"35","author":[{"given":"Daeun","family":"Noh","sequence":"first","affiliation":[{"name":"Department of Chemistry The University of Texas at Austin  Austin Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2020-2641","authenticated-orcid":false,"given":"Robert 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