Rational stabilization of complex proteins: a divide and combine approach

Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velazquez-Campoy, Adrian; Sancho, Javier

Abstract

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 degrees 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).

Más información

Título según WOS: ID WOS:000351180900003 Not found in local WOS DB
Título de la Revista: SCIENTIFIC REPORTS
Volumen: 5
Editorial: NATURE PORTFOLIO
Fecha de publicación: 2015
DOI:

10.1038/srep09129

Notas: ISI