48440 20210121114541.0 doi 10.1038/srep09129 sideral 93811 ART-2015-93811 eng Llamazares, Emilio Rational stabilization of complex proteins: A divide and combine approach 2015 Access copy available to the general public Unrestricted Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). info:eu-repo/grantAgreement/ES/DGA/B89 info:eu-repo/grantAgreement/ES/MICINN/BFU2010-16297 info:eu-repo/grantAgreement/ES/MINECO/BFU2013-47064-P info:eu-repo/semantics/openAccess by http://creativecommons.org/licenses/by/3.0/es/ 5.228 2015 MULTIDISCIPLINARY SCIENCES 7 / 62 = 0.113 2015 Q1 T1 2.034 2015 Multidisciplinary 2015 Q1 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion (orcid)0000-0003-4597-4020 Clemente, Isabel Universidad de Zaragoza Bueno, Marta (orcid)0000-0001-5702-4538 Velázquez-Campoy, Adrián Universidad de Zaragoza (orcid)0000-0002-2879-9200 Sancho, Javier Universidad de Zaragoza 2009 750 Universidad de Zaragoza Dpto. Química Analítica Área Química Analítica 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 5 (2015), 9129 [11 pp.] Sci. rep. Scientific Reports 2045-2322 1984482 http://zaguan.unizar.es/record/48440/files/texto_completo.pdf Versión publicada 102771 http://zaguan.unizar.es/record/48440/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:48440 articulos driver 2021-01-21-11:16:29 ARTICLE