Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Endolysins are bacteriophage-derived enzymes with therapeutic potential.
• Streptococcal endolysin PlyC shows efficacy but suffers from poor thermal stability.
• Limited stability hinders the development of endolysins as antimicrobials.

Purpose of the Study:

• To enhance the thermal stability of the streptococcal endolysin PlyC.
• To engineer a more robust enzyme for potential antimicrobial applications.
• To identify specific mutations that improve PlyC's stability.

Main Methods:

• Computational protein engineering using FoldX and Rosetta algorithms.
• Site-directed mutagenesis to create PlyC point mutants.
• Thermal characterization of wild-type and mutant PlyC enzymes.

Main Results:

• One mutant, PlyC (PlyCA) T406R, exhibited a 2.2°C increase in thermal denaturation temperature.
• The T406R mutation resulted in a 16-fold increase in kinetic stability compared to wild type.
• Computational predictions of stabilizing mutations did not fully correlate with experimental results.

Conclusions:

• The PlyC T406R mutation significantly enhances enzyme stability, addressing a key limitation for therapeutic use.
• Protein engineering can improve the stability of bacteriophage endolysins.
• Further investigation into the structure-stability relationship of PlyC is warranted.