Rational stabilization of complex proteins: a divide and combine approach

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• Protein thermostability is critical for applications in analytics, synthesis, and therapeutics.
• Existing strategies for protein stabilization often fail for large, non-cooperative proteins.

Purpose of the Study:

• To develop a novel strategy for enhancing the thermostability of large, non-fully cooperative proteins.
• To identify and target regions of lower stability within complex proteins for rational engineering.

Main Methods:

• Engineering apoflavodoxin, a known non-cooperative protein, using structure-based design.
• Step-wise introduction of rationally designed mutations focused on less stable regions.
• Analysis of thermal unfolding cooperative behavior using van't Hoff to calorimetric enthalpy ratios.

Main Results:

• Achieved fully-cooperative thermal unfolding in engineered apoflavodoxin variants.
• Increased the melting temperature by 32°C, reaching 75°C.
• Demonstrated successful stabilization of a previously challenging protein.

Conclusions:

• Targeting regions of lower stability is key to stabilizing complex, non-cooperative proteins.
• The presented strategy offers a promising approach for thermostabilization of diverse complex proteins.
• Findings may inform formulation strategies and the use of stabilizing ligands like pharmacological chaperones.