000163800 001__ 163800
000163800 005__ 20251107115328.0
000163800 0247_ $$2doi$$a10.1016/j.ijbiomac.2025.148333
000163800 0248_ $$2sideral$$a145860
000163800 037__ $$aART-2025-145860
000163800 041__ $$aeng
000163800 100__ $$aHidalgo-Toledo, Antonio$$uUniversidad de Zaragoza
000163800 245__ $$aProtein thermostabilization with Protposer: Pushing the stability limits and folding reversibility of a highly-stabilized apoflavodoxin
000163800 260__ $$c2025
000163800 5060_ $$aAccess copy available to the general public$$fUnrestricted
000163800 5203_ $$aEnhancing the stability of highly stable proteins represents an interesting challenge in protein design. We have used the computational tool Protposer to rapidly achieve large additional stabilization of apoflavodoxin, a protein strongly thermostabilized over the years through protein engineering based on educated guesses. By rationally combining top-ranked mutations onto a previously stabilized variant (6 M), we have generated a series of new mutants and characterized their stability by thermal and chemical denaturation. Relative to the starting variant, the Tm of 10 M apoflavodoxin is nearly 9 °C higher, while the simplified 3 M and 4 M mutants, showing improved refolding properties, display increases of 6/7.5 °C, respectively. The thermostabilizing effects of individual mutations are close to additive and translate into a large increase in conformational stability at 25.0 °C. Comparison of the x-ray structures of progressively stabilized WT, 6 M and 10 M flavodoxins reveals a concomitant mild trend toward shorter hydrogen bonds, reduced internal cavity volumes and narrower tunnels. Overall, these conformational changes are minor, and a functional assay confirms the mutants also preserve their catalytic activity. These findings demonstrate that even highly stable proteins can be further stabilized through rational design using a simple computational tool that automatically analyses PDB files and identifies stabilizing mutations.
000163800 536__ $$9info:eu-repo/grantAgreement/ES/DGA/B23-24$$9info:eu-repo/grantAgreement/ES/DGA/E45-23R$$9info:eu-repo/grantAgreement/EC/H2020/101004806/EU/MOlecular-Scale Biophysics Research Infrastructure/MOSBRI$$9This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 101004806-MOSBRI$$9info:eu-repo/grantAgreement/ES/MICINN/PID2022-141068NB-I00
000163800 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc$$uhttps://creativecommons.org/licenses/by-nc/4.0/deed.es
000163800 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000163800 700__ $$aBazco, Darío
000163800 700__ $$aCorrea-Pérez, Víctor$$uUniversidad de Zaragoza
000163800 700__ $$0(orcid)0000-0001-9047-0046$$aMartínez-Júlvez, Marta$$uUniversidad de Zaragoza
000163800 700__ $$0(orcid)0000-0002-2879-9200$$aSancho, Javier$$uUniversidad de Zaragoza
000163800 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000163800 773__ $$g331 (2025), 148333 [11 pp.]$$pInt. j. biol. macromol.$$tInternational journal of biological macromolecules$$x0141-8130
000163800 8564_ $$s3635212$$uhttps://zaguan.unizar.es/record/163800/files/texto_completo.pdf$$yVersión publicada
000163800 8564_ $$s2687954$$uhttps://zaguan.unizar.es/record/163800/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000163800 909CO $$ooai:zaguan.unizar.es:163800$$particulos$$pdriver
000163800 951__ $$a2025-11-07-10:24:57
000163800 980__ $$aARTICLE