Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split...

Area of Science:

• Biochemistry
• Molecular Biology
• Bioengineering

Background:

• Click beetle luciferase (CBLuc) offers stable, red light emission optimal for bioanalytical signaling.
• Studying signal-controlled protein-protein interactions is crucial for understanding cellular processes.

Purpose of the Study:

• To validate a single-molecule-format complementation system using split CBLuc.
• To investigate signal-controlled protein-peptide interactions using this novel probe.

Main Methods:

• Generated 10 pairs of N- and C-terminal CBLuc fragments for intramolecular complementation.
• Fused androgen receptor ligand-binding domain (AR LBD) to a peptide, linking it to split CBLuc fragments.
• Utilized androgen to induce protein association and restore CBLuc activity.

Main Results:

• Identified optimal CBLuc dissection sites (D412 and I439) for stable activity recovery.
• Demonstrated ligand sensitivity and kinetics of the split CBLuc probe in various cell lines.
• Validated the probe in different protein-peptide binding models.

Conclusions:

• The split CBLuc system effectively studies signal-controlled protein-peptide interactions.
• The probe is applicable for developing androgen receptor (AR) signaling therapeutics.
• This system can screen chemicals affecting protein signal transduction in vivo.