doi:10.1016/j.ijbiomac.2025.148333engHidalgo-Toledo, AntonioBazco, DaríoCorrea-Pérez, VíctorMartínez-Júlvez, MartaSancho, JavierProtein thermostabilization with Protposer: Pushing the stability limits and folding reversibility of a highly-stabilized apoflavodoxinART-2025-145860Enhancing 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.2025http://zaguan.unizar.es/record/16380010.1016/j.ijbiomac.2025.148333http://zaguan.unizar.es/record/163800oai:zaguan.unizar.es:163800info:eu-repo/grantAgreement/ES/DGA/B23-24info:eu-repo/grantAgreement/ES/DGA/E45-23Rinfo:eu-repo/grantAgreement/EC/H2020/101004806/EU/MOlecular-Scale Biophysics Research Infrastructure/MOSBRIThis project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 101004806-MOSBRIinfo:eu-repo/grantAgreement/ES/MICINN/PID2022-141068NB-I00International journal of biological macromolecules 331 (2025), 148333 [11 pp.]by-nchttps://creativecommons.org/licenses/by-nc/4.0/deed.esinfo:eu-repo/semantics/openAccess