Rational stabilization of complex proteins: A divide and combine approach

Llamazares, Emilio ; Clemente, Isabel (Universidad de Zaragoza) ; Bueno, Marta ; Velázquez-Campoy, Adrián (Universidad de Zaragoza) ; Sancho, Javier (Universidad de Zaragoza)

Resumen: 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).

Idioma: Inglés

DOI: 10.1038/srep09129

Año: 2015

Publicado en: Scientific Reports 5 (2015), 9129 [11 pp.]
ISSN: 2045-2322

Factor impacto JCR: 5.228 (2015)

Categ. JCR: MULTIDISCIPLINARY SCIENCES rank: 7 / 62 = 0.113 (2015) - Q1 - T1

Factor impacto SCIMAGO: 2.034 - Multidisciplinary (Q1)

Financiación: info:eu-repo/grantAgreement/ES/DGA/B89
Financiación: info:eu-repo/grantAgreement/ES/MICINN/BFU2010-16297
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BFU2013-47064-P

Tipo y forma: Article (Published version)

Área (Departamento): Área Química Analítica (Dpto. Química Analítica)
Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)