Rational stabilization of complex proteins: a divide and combine approach

Scientific Reports
Emilio LamazaresJavier Sancho

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°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).

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Citations

Jun 13, 2015·International Journal of Molecular Sciences·Enrico MalitoMatthew J Bottomley
Jul 14, 2017·Physical Chemistry Chemical Physics : PCCP·Emilio LamazaresJavier Sancho
Aug 18, 2017·Molecular BioSystems·Andrew J OlsenJin Kim Montclare
Jul 3, 2021·International Journal of Molecular Sciences·María Conde-Giménez, Javier Sancho

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Methods Mentioned

BETA
differential scanning calorimetry
Fluorescence
NMR
protein folding

Software Mentioned

MLAB
Origin
ProtSA

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