Structure-guided engineering of protein stability through core hydrophobicity

Area of Science:

• Protein Engineering
• Biophysics
• Computational Biology

Background:

• Protein thermostability is crucial for industrial and biomedical applications, enhancing integrity and function at high temperatures.
• Current methods for improving protein stability are limited, necessitating novel strategies.

Purpose of the Study:

• To develop a computational method for enhancing protein thermal stability by optimizing the hydrophobic core.
• To validate the method's efficacy using experimental and simulation techniques.

Main Methods:

• Developed an algorithm to identify and substitute buried hydrophobic residues with longer or bulkier side chains.
• Calculated free energy of unfolding (ΔG) to select stabilizing substitutions, excluding functionally critical regions.
• Applied the method to beta-grasp proteins, with experimental validation on NEDD8.

Main Results:

• Two predicted substitutions significantly increased NEDD8's thermal stability (1.7 kcal/mol) and melting point (17°C).
• Molecular dynamics (MD) simulations and NMR spectroscopy showed reduced fluctuations and enhanced stabilizing interactions.
• Functional assays confirmed that engineered substitutions preserved NEDD8's fold and biological activity.

Conclusions:

• Optimizing buried hydrophobic residues is an effective strategy for enhancing protein thermal stability.
• The developed computational approach provides a general framework for designing robust proteins for various applications.