Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• The Par-4 protein is linked to various diseases, including cancers and neurodegenerative disorders.
• The Par-4 C-terminus peptide unfolds at neutral pH and forms a coiled coil at acidic pH.
• This peptide mediates Par-4 self-association and interactions with effector molecules.

Purpose of the Study:

• To investigate the role of specific acidic residues in the pH-dependent folding of the Par-4 C-terminus peptide.
• To understand the mechanism of coiled coil formation in the Par-4 C-terminus.

Main Methods:

• Site-directed mutagenesis of two acidic residues in the Par-4 C-terminus peptide.
• Circular dichroism (CD) spectroscopy to analyze peptide folding and unfolding.
• Investigating the effects of pH and temperature on peptide conformation.

Main Results:

• Mutating the two acidic residues significantly altered the peptide's folding/unfolding equilibrium.
• This confirms the critical role of these residues in pH-dependent folding.
• The findings suggest charge repulsion at the coiled coil interface influences folding.

Conclusions:

• The two acidic residues are key determinants of the Par-4 C-terminus coiled coil's pH-dependent folding.
• Alleviating charge repulsion, via low pH or complementary electrostatic environments, is crucial for efficient folding.
• This provides insights into the structural regulation of Par-4 function in disease.