The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach

Area of Science:

• Biochemistry and structural biology
• Protein engineering and design

Background:

• Designing stable proteins with specific functions is a key goal in biochemistry.
• Understanding protein stability determinants is crucial for expanding the applications of de novo proteins.
• The (β/α)8-barrel (TIM-barrel) fold is a common and versatile protein scaffold.

Purpose of the Study:

• To design and analyze a collection of stable de novo TIM barrels (DeNovoTIMs).
• To explore the stability landscape of engineered proteins.
• To investigate the molecular basis of epistasis in designed proteins.

Main Methods:

• Computational fixed-backbone and modular protein design approach.
• Improved hydrophobic packing strategies based on the sTIM11 scaffold.
• Thorough folding and stability analysis of designed DeNovoTIMs.

Main Results:

• Successfully designed stable DeNovoTIMs with significant variations in melting temperature (up to 60°C) and conformational stability (up to 22 kcal/mol).
• Observed significant non-additive (epistatic) effects when combining stabilizing mutations.
• Identified the extension of hydrophobic cores as a potential molecular basis for epistasis in DeNovoTIMs.

Conclusions:

• DeNovoTIMs represent a novel class of proteins exploring uncharted regions of the protein stability landscape.
• The study demonstrates the potential for fine-tuning protein stability through rational design.
• Understanding epistasis is critical for the successful engineering of complex protein functions.