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The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.<\/jats:p>","DOI":"10.3390\/toxins16030133","type":"journal-article","created":{"date-parts":[[2024,3,4]],"date-time":"2024-03-04T04:36:21Z","timestamp":1709526981000},"page":"133","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-0781-820X","authenticated-orcid":false,"given":"Cheuk W.","family":"Au","sequence":"first","affiliation":[{"name":"School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3765-0325","authenticated-orcid":false,"given":"Iain","family":"Manfield","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK"},{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael E.","family":"Webb","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"},{"name":"School of Chemistry, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emanuele","family":"Paci","sequence":"additional","affiliation":[{"name":"Dipartimento di Fisica e Astronomia \u201cAugusto Righi\u201d, Viale Berti Pichat 6\/2, 40127 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7352-0360","authenticated-orcid":false,"given":"W. Bruce","family":"Turnbull","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"},{"name":"School of Chemistry, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4784-103X","authenticated-orcid":false,"given":"James F.","family":"Ross","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK"},{"name":"Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"508","DOI":"10.3109\/1061186X.2015.1095194","article-title":"Methods to Follow Intracellular Trafficking of Cell-Penetrating Peptides","volume":"24","author":"Arukuusk","year":"2016","journal-title":"J. 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