Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3

Area of Science:

• Protein biochemistry
• Molecular biology
• Biophysics

Background:

• Post-synaptic density-95 (PSD95) protein's third PDZ domain (PSD95-PDZ3) exhibits three-state thermal denaturation and amyloidogenic properties.
• The intermediate state of PSD95-PDZ3 involves reversible oligomerization (RO).
• A previously identified F340A mutation inhibits RO and amyloidogenesis.

Purpose of the Study:

• To engineer PDZ3 variants that induce high-temperature RO and amyloidogenesis.
• To investigate the role of specific hydrophilic residues in protein aggregation.
• To understand the structural basis of amyloid formation.

Main Methods:

• Site-directed mutagenesis to create three PDZ3-F340A variants (R309L, E310L, N326L).
• Differential scanning calorimetry (DSC) to analyze thermal denaturation.
• Circular dichroism (CD) spectroscopy to assess secondary structure changes.
• Thioflavin T (ThT) fluorescence assay to detect amyloid formation.

Main Results:

• Two variants (PDZ3-F340A/R309L and E310L) showed two-state denaturation.
• PDZ3-F340A/N326L exhibited three-state denaturation, high-temperature RO, and amyloidogenicity.
• Secondary structures of PDZ3-F340A/N326L and wild-type in RO state were unfolded.
• A single mutation (N326L) was sufficient to trigger high-temperature RO and amyloidogenesis.

Conclusions:

• Single amino acid substitutions can reverse-engineer protein behavior to induce aggregation.
• Hydrophobic mutations in specific surface-exposed residues can promote high-temperature RO and amyloidogenesis.
• This work provides a model for studying the initiation of protein misfolding and aggregation pathways.