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Thermostable proteins can be unfolded by combined chemical and thermal denaturation followed by global analysis of multiple melting curves. Here, we have developed CheMelt, a new online tool for global analysis of unfolding data via an intuitive graphical user interface. We use nanoscale differential scanning fluorimetry followed by CheMelt data analysis to dissect the combined thermal and chemical denaturation of 35 de novo designed protein binders. Thirteen present sufficient fluorescence changes to extract thermodynamic parameters of unfolding. These de novo designed proteins have systematically lower \u0394\n                    <jats:italic>C<\/jats:italic>\n                    <jats:sub>p<\/jats:sub>\n                    and\n                    <jats:italic>m<\/jats:italic>\n                    \u2010values than comparable natural proteins. We show that a high thermostability of a designed protein does not necessarily imply a high equilibrium stability, and demonstrate the potential of CheMelt in dissecting thermodynamic properties for protein design and engineering. CheMelt can be accessed at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"https:\/\/spc.embl-hamburg.de\/app\/chemelt\">https:\/\/spc.embl-hamburg.de\/app\/chemelt<\/jats:ext-link>\n                    .\n                  <\/jats:p>","DOI":"10.1002\/pro.70800","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T11:20:01Z","timestamp":1790767201000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Global analysis of thermal and chemical denaturation using\n                    <scp>CheMelt<\/scp>\n                    : Thermodynamic dissection of highly thermostable de novo designed proteins"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2153-6149","authenticated-orcid":false,"given":"Vili","family":"Lampinen","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and 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Aarhus Denmark"},{"name":"The Danish Research Institute of Translational Neuroscience (DANDRITE) Aarhus University  Aarhus Denmark"},{"name":"The Center for Proteins in Memory (PROMEMO) Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Florian","family":"V\u00f6gele","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory Hamburg  Hamburg Germany"},{"name":"Centre for Structural Systems Biology  Hamburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3778-1528","authenticated-orcid":false,"given":"Francisca","family":"Pinheiro","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics Aarhus University  Aarhus Denmark"},{"name":"Interdisciplinary Nanoscience Center (iNANO) Aarhus University  Aarhus Denmark"},{"name":"The Danish Research Institute of Translational Neuroscience (DANDRITE) Aarhus University  Aarhus 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