Thermostabilizing mutations preferentially occur at structural weak spots with a high mutation ratio.

Area of Science:

• Biophysics
• Structural Biology
• Computational Biology

Background:

• Thermostability is crucial for protein function in varying environments.
• Understanding the link between protein structure and thermostability is key for protein engineering.
• Citrate synthase (CS) serves as a model system to study protein thermostability.

Purpose of the Study:

• To investigate the relationship between structural rigidity and thermostability of citrate synthase (CS) using Constraint Network Analysis (CNA).
• To introduce and apply an ensemble-based variant of CNA, incorporating temperature-dependent hydrophobic interactions.
• To validate CNA's ability to quantitatively discriminate between proteins of differing thermostability.

Main Methods:

• Application of Constraint Network Analysis (CNA) to five CS structures across a temperature range (37 °C to 100 °C).
• Development of an ensemble-based CNA variant to model temperature-dependent hydrophobic interactions.
• Correlation analysis between predicted CS thermostabilities and optimal growth temperatures of source organisms.

Main Results:

• A strong correlation (R²=0.88, p=0.017) was found between predicted CS thermostabilities and organism growth temperatures.
• CNA successfully identified structural weak spots in less thermostable CS, which showed higher mutation ratios in more stable CS.
• Atomic-level analysis revealed that thermophilic CS utilize enhanced hydrogen bonding, while hyperthermophilic CS incorporate more hydrophobic contacts for thermostability.

Conclusions:

• CNA is a validated method for quantitatively assessing protein thermostability, even among orthologs.
• Structural weak spots identified by CNA are critical targets for mutations that enhance thermostability.
• CNA can serve as a pre-filter in protein engineering to identify residues likely to improve thermostability upon mutation.