Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• Protein thermostability and proteolytic resistance are often correlated.
• The structural basis for this association, particularly the role of loops, requires further investigation.
• Understanding this relationship can guide protein engineering efforts.

Purpose of the Study:

• To investigate the relationship between protein global stability and proteolytic resistance.
• To identify specific mutations that enhance both properties in a model protein.
• To determine if proteolytic resistance correlates with global stability across a range of single-point mutants.

Main Methods:

• Loop scanning site-saturation mutagenesis was performed on Bacillus subtilis lipase at 86 positions.
• ~16,000 clones were screened to identify mutants with improved thermostability.
• Thermostability (apparent melting temperature, Tm(app)) and proteolytic resistance (residual activity after incubation with Subtilisin A) were measured for selected mutants.

Main Results:

• Seventeen single mutants with increased thermostability (1-6°C) and free energy of unfolding (ΔG(unf)) were identified.
• A strong positive correlation (r > 0.9) was observed between proteolytic resistance and ΔG(unf).
• This correlation held true even with minimal sequence changes, covering a significant portion of the protein surface.

Conclusions:

• Proteolytic resistance is strongly correlated with the global stability of Bacillus subtilis lipase.
• This relationship is robust and observable even with minor sequence modifications.
• The findings support the covariance of protein stability and resistance to proteolysis, offering insights for protein design.