Au, C.W. orcid.org/0009-0007-0781-820X, Manfield, I., Webb, M.E. et al. (3 more authors) (2024) The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity. Toxins, 16 (3). 133. ISSN 2072-6651
Abstract
Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. 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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | cholera toxin B-subunit; CTB; GM1 ganglioside; melting temperature; dissociation constant; Rosetta; mutational-space map; differential scanning fluorimetry; isothermal titration calorimetry |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 05 Mar 2024 11:15 |
Last Modified: | 23 Jan 2025 11:03 |
Status: | Published |
Publisher: | MDPI |
Identification Number: | 10.3390/toxins16030133 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:209910 |