LuBiA (Luciferase-Based Binding Assay): Glowing Peptides as Sensitive Probes to Study Ligand-Receptor Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Quantitative and qualitative biochemical descriptions of molecular interactions are crucial for understanding ligand/receptor pairs and their structure/function.
• Bioactive peptides, often active at (sub-)nanomolar concentrations, exhibit high affinity for receptor binding sites.
• The low abundance of receptor proteins necessitates highly sensitive detection methods for studying ligand/receptor interactions.

Purpose of the Study:

• To present a protocol for an inexpensive luminescence-based detection setup.
• To enable sensitive quantification of peptide ligands and study of their interactions with receptors.

Main Methods:

• The peptide ligand of interest is extended with the 11-amino acid HiBiT tag.
• The HiBiT tag facilitates the reconstitution of enzymatic activity with LgBiT, a truncated Oplophorus gracilirostris luciferase.
• Luminescence detection quantifies the reconstituted enzymatic activity, allowing for sensitive measurement of the tagged peptide.

Main Results:

• The developed protocol allows for the quantification of the HiBiT-tagged peptide ligand.
• The method achieves quantification down to femtomole (fmol) amounts.
• This luminescence-based setup provides a sensitive and cost-effective means to study ligand/receptor interactions.

Conclusions:

• A simple, inexpensive, and highly sensitive luminescence-based detection protocol for peptide ligands has been established.
• The HiBiT/LgBiT system enables precise quantification of low-abundance peptides, facilitating the study of their interactions with receptors.
• This method is valuable for investigating ligand/receptor pairs and their structure/function relationships in molecular biology and biochemistry.