doi:10.1038/srep09129engLlamazares, EmilioClemente, IsabelBueno, MartaVelázquez-Campoy, AdriánSancho, JavierRational stabilization of complex proteins: A divide and combine approachART-2015-93811Increasing 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).2015http://zaguan.unizar.es/record/4844010.1038/srep09129http://zaguan.unizar.es/record/48440oai:zaguan.unizar.es:48440info:eu-repo/grantAgreement/ES/DGA/B89info:eu-repo/grantAgreement/ES/MICINN/BFU2010-16297info:eu-repo/grantAgreement/ES/MINECO/BFU2013-47064-PScientific Reports 5 (2015), 9129 [11 pp.]byhttp://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccess