Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor

Area of Science:

• Protein Engineering
• Biochemistry
• Molecular Biology

Background:

• Bacteriophage P22 Arc repressor is a dimeric protein that regulates gene expression.
• Understanding protein folding and stability is crucial for protein engineering and drug design.
• Single-chain variants can offer insights into protein-protein interactions and folding pathways.

Purpose of the Study:

• To investigate the structural and functional properties of a single-chain variant of the Arc repressor (Arc-L1-Arc).
• To compare the folding and unfolding thermodynamics and kinetics of Arc-L1-Arc with the native Arc dimer.
• To determine the effect of a peptide linker on the stability and refolding behavior of the Arc repressor.

Main Methods:

• Spectroscopic probes (fluorescence, circular dichroism) were used to monitor protein structure.
• Equilibrium denaturation experiments were performed using urea gradients.
• Kinetic refolding and unfolding experiments were conducted to analyze reaction rates.
• Two-state model analysis was applied to denaturation isotherms.

Main Results:

• Arc-L1-Arc exhibits native and denatured states similar to the unlinked Arc dimer.
• Denaturation follows a two-state model without populated intermediates.
• The effective subunit concentration in the denatured state was determined to be 2.7 mM.
• Refolding kinetics indicate a significantly higher effective subunit concentration (4.5 mM) due to the linker.
• Unfolding rate is faster in Arc-L1-Arc, while refolding is significantly enhanced.

Conclusions:

• The 15-residue linker in Arc-L1-Arc does not significantly disrupt the native structure but enhances refolding efficiency.
• Arc-L1-Arc serves as a valuable model for studying intramolecular folding pathways and effective concentrations.
• The study provides insights into the role of covalent linkage in protein stability and folding dynamics.