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In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed <jats:italic>Escherichia coli<\/jats:italic> and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine <jats:italic>N<\/jats:italic>-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.<\/jats:p>","DOI":"10.1038\/s41589-024-01568-7","type":"journal-article","created":{"date-parts":[[2024,2,29]],"date-time":"2024-02-29T11:02:17Z","timestamp":1709204537000},"page":"1053-1065","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":55,"title":["In situ analysis of osmolyte mechanisms of proteome thermal stabilization"],"prefix":"10.1038","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8502-1772","authenticated-orcid":false,"given":"Monika","family":"Pepelnjak","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8397-3515","authenticated-orcid":false,"given":"Britta","family":"Velten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6845-7400","authenticated-orcid":false,"given":"Nicolas","family":"N\u00e4pflin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatjana","family":"von Rosen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Umberto Capasso","family":"Palmiero","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2000-3789","authenticated-orcid":false,"given":"Jeong Hoon","family":"Ko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3692-6289","authenticated-orcid":false,"given":"Heather D.","family":"Maynard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2740-1205","authenticated-orcid":false,"given":"Paolo","family":"Arosio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5773-9274","authenticated-orcid":false,"given":"Eilika","family":"Weber-Ban","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie","family":"de Souza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0474-2218","authenticated-orcid":false,"given":"Wolfgang","family":"Huber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4109-3552","authenticated-orcid":false,"given":"Paola","family":"Picotti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,29]]},"reference":[{"key":"1568_CR1","doi-asserted-by":"crossref","first-page":"2559","DOI":"10.1021\/pr3000128","volume":"11","author":"Y Ye","year":"2012","unstructured":"Ye, Y. et al. 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