Budded baculoviruses as a receptor display system to quantify ligand binding with TIRF microscopy

Area of Science:

• Biophysics
• Biochemistry
• Microscopy

Background:

• Studying receptor-ligand interactions is crucial for drug discovery but limited by current measurement techniques.
• Existing methods often compromise on receptor environment, throughput, or sensitivity.

Purpose of the Study:

• To develop a sensitive, high-throughput microscopy method for studying receptor-ligand interactions.
• To enable detailed analysis of ligand binding within a natural receptor environment.

Main Methods:

• Utilized total internal reflection fluorescence microscopy (TIR-FM).
• Combined G protein-coupled receptor (GPCR) display on baculovirus with functionalized coverslip immobilization.
• Developed open-source multiwell systems and SPOTNIC image analysis software.

Main Results:

• Successfully validated selective immobilization of baculovirus particles displaying Frizzled 6 and neuropeptide Y Y1 receptors.
• Determined ligand affinities for the neuropeptide Y Y1 receptor, consistent with existing data and parallel fluorescence anisotropy measurements.
• Demonstrated the method's scalability and flexibility for various ligand binding assays.

Conclusions:

• The novel TIR-FM method provides a balanced approach for receptor-ligand interaction studies.
• This technique enhances sensitivity, throughput, and preserves the natural lipid environment of receptors.
• The method is adaptable for advanced assays, including super-resolution and kinetic measurements.