Repacking the Core of T4 lysozyme by automated design

Area of Science:

• Protein Engineering
• Computational Biology
• Structural Biology

Background:

• Protein core stability is crucial for function.
• Automated protein redesign offers a computational approach to engineer protein stability and properties.
• T4 lysozyme is a model system for studying protein stability and redesign.

Purpose of the Study:

• To redesign the core of phage T4 lysozyme using automated computational methods.
• To investigate the structural and thermodynamic consequences of core redesign.
• To assess the effectiveness of incorporating penalties for specific residues, like methionine, during redesign.

Main Methods:

• Utilized the ORBIT program for automated protein redesign of T4 lysozyme's C-terminal domain.
• Allowed variations in sequence and side-chain conformation at buried sites while fixing the backbone.
• Performed crystallographic and thermal analyses on designed variants (Core-7, Core-10) and mutants.

Main Results:

• Redesigned proteins (Core-7, Core-10) showed some achieved repacking but were not more stable than wild-type.
• A methionine penalty effectively influenced the redesign outcome (Core-10 had ten substitutions).
• Observed side-chain conformations in redesigned proteins often retained native-like rotamers, and backbone shifts occurred.

Conclusions:

• Protein core redesign can achieve local repacking but does not guarantee enhanced stability.
• Side-chain packing in protein cores is specific, and destabilization often results from substitutions.
• Future redesign strategies may need to account for backbone flexibility and the role of internal water molecules.