Stabilization of phage T4 lysozyme by engineered disulfide bonds

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• T4 lysozyme is a model system for studying protein stability.
• Disulfide bonds can stabilize protein structures.
• Previous studies have explored disulfide bond introduction for protein stabilization.

Purpose of the Study:

• To engineer disulfide bridges into cysteine-free T4 lysozyme.
• To assess the impact of these engineered disulfide bonds on thermal stability.
• To understand the relationship between disulfide bridge characteristics and protein stability.

Main Methods:

• Computational modeling to predict suitable sites for disulfide bond introduction.
• Site-directed mutagenesis to introduce cysteine residues.
• In vitro oxidation to form disulfide bonds.
• Thermal denaturation assays (monitoring melting temperature) to measure stability.
• Equilibrium measurements of disulfide bond reduction to assess crosslink favorability.

Main Results:

• Four disulfide bridges were successfully engineered into T4 lysozyme.
• All oxidized (crosslinked) lysozymes exhibited increased stability compared to reduced forms.
• Two mutants (9-164 and 21-142) showed significant increases in melting temperature (6.4°C and 11.0°C, respectively).
• Other mutants displayed less or equal stability, correlating with less favorable crosslinks in the native structure.
• Effective stabilizing crosslinks were associated with large loop sizes and flexible molecular regions.

Conclusions:

• Engineered disulfide bonds can enhance the thermal stability of T4 lysozyme.
• The effectiveness of disulfide bridges depends on their location, loop size, and the flexibility of the protein structure.
• Stabilization is a balance between favorable entropic effects in the unfolded state and strain energy in the folded state.
• Protein flexibility is a key consideration for designing stabilizing disulfide bridges.