Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds

Area of Science:

• Biomolecular engineering
• Protein engineering
• Biocatalysis

Background:

• Protein stability is crucial for biomolecular engineering and therapeutic design.
• T4 lysozyme is a model enzyme used in protein studies.
• Unnatural amino acids offer novel ways to modify protein properties.

Purpose of the Study:

• To enhance the thermal stability and enzymatic activity of T4 lysozyme.
• To investigate the role of halogen bonding in protein stabilization.
• To explore the application of unnatural amino acids in protein design.

Main Methods:

• Site-directed mutagenesis to incorporate m-chlorotyrosine (^mClY) into T4 lysozyme.
• Thermal stability assays (melting temperature and enthalpy).
• Enzymatic activity assays at elevated temperatures.
• Quantum chemical calculations to analyze bonding interactions.

Main Results:

• ^mClY incorporation increased T4 lysozyme's melting temperature by ~1 °C and melting enthalpy by 3 kcal/mol.
• Enzymatic activity at 40 °C was 15% higher compared to the wild-type enzyme.
• A novel hydrogen bond-enhanced halogen bond (HeX-B) interaction was identified between ^mClY and glycine 28.
• Larger halogens (bromine, iodine) did not confer similar stability or activity enhancements.

Conclusions:

• Engineered halogen bonds, specifically HeX-B, can effectively stabilize enzymes and enhance their activity.
• Unnatural amino acids like m-chlorotyrosine are valuable tools for designing more stable protein therapeutics.
• This study demonstrates a new strategy for protein engineering using halogenated amino acids.