Disulfide bonds and thermal stability in T4 lysozyme

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Disulfide bonds are crucial for stabilizing extracellular proteins, but their precise mechanisms remain unclear.
• The role of disulfide bonds in protein thermal stability, particularly against irreversible inactivation, requires further investigation.

Purpose of the Study:

• To investigate the relationship between the thermodynamic contribution of an engineered disulfide bond to protein folding and its effect on irreversible thermal inactivation.
• To elucidate the mechanisms by which disulfide bonds confer stability to proteins like T4 lysozyme.

Main Methods:

• Thermodynamic analysis of wild-type and mutant T4 lysozymes (non-crosslinked and crosslinked variants).
• Assessment of protein activity loss and recovery after thermal stress and denaturation/renaturation treatments.
• Construction and analysis of multiple mutants combining the disulfide bond with temperature-sensitive lesions.

Main Results:

• An engineered 3-97 disulfide bond stabilizes T4 lysozyme against both reversible unfolding and irreversible thermal inactivation.
• Non-crosslinked T4 lysozyme is highly sensitive to irreversible inactivation above its melting temperature, with activity recoverable by denaturation/renaturation.
• Crosslinked T4 lysozyme exhibits slower inactivation rates, independent of conformational changes, suggesting chemical inactivation pathways.

Conclusions:

• The 3-97 disulfide bond enhances stability against irreversible inactivation through a mechanism largely independent of its thermodynamic contribution to folding.
• Disulfide bonds may stabilize proteins by restricting unfolded states to more compact conformations, reducing hydrophobic surface exposure.
• Findings have implications for protein engineering strategies utilizing disulfide bonds and understanding their evolutionary roles.