163800 20251107115328.0 doi 10.1016/j.ijbiomac.2025.148333 sideral 145860 ART-2025-145860 eng Hidalgo-Toledo, Antonio Universidad de Zaragoza Protein thermostabilization with Protposer: Pushing the stability limits and folding reversibility of a highly-stabilized apoflavodoxin 2025 Enhancing the stability of highly stable proteins represents an interesting challenge in protein design. We have used the computational tool Protposer to rapidly achieve large additional stabilization of apoflavodoxin, a protein strongly thermostabilized over the years through protein engineering based on educated guesses. By rationally combining top-ranked mutations onto a previously stabilized variant (6 M), we have generated a series of new mutants and characterized their stability by thermal and chemical denaturation. Relative to the starting variant, the Tm of 10 M apoflavodoxin is nearly 9 °C higher, while the simplified 3 M and 4 M mutants, showing improved refolding properties, display increases of 6/7.5 °C, respectively. The thermostabilizing effects of individual mutations are close to additive and translate into a large increase in conformational stability at 25.0 °C. Comparison of the x-ray structures of progressively stabilized WT, 6 M and 10 M flavodoxins reveals a concomitant mild trend toward shorter hydrogen bonds, reduced internal cavity volumes and narrower tunnels. Overall, these conformational changes are minor, and a functional assay confirms the mutants also preserve their catalytic activity. These findings demonstrate that even highly stable proteins can be further stabilized through rational design using a simple computational tool that automatically analyses PDB files and identifies stabilizing mutations. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/DGA/B23-24 info:eu-repo/grantAgreement/ES/DGA/E45-23R info:eu-repo/grantAgreement/EC/H2020/101004806/EU/MOlecular-Scale Biophysics Research Infrastructure/MOSBRI This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 101004806-MOSBRI info:eu-repo/grantAgreement/ES/MICINN/PID2022-141068NB-I00 info:eu-repo/semantics/openAccess by-nc https://creativecommons.org/licenses/by-nc/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Bazco, Darío Correa-Pérez, Víctor Universidad de Zaragoza Martínez-Júlvez, Marta Universidad de Zaragoza (orcid)0000-0001-9047-0046 Sancho, Javier Universidad de Zaragoza (orcid)0000-0002-2879-9200 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 331 (2025), 148333 [11 pp.] Int. j. biol. macromol. International journal of biological macromolecules 0141-8130 3635212 http://zaguan.unizar.es/record/163800/files/texto_completo.pdf Versión publicada 2687954 http://zaguan.unizar.es/record/163800/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:163800 articulos driver 2025-11-07-10:24:57 ARTICLE