Label-free Quantification of Direct Protein-protein Interactions with Backscattering Interferometry

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Cellular function relies on precise macromolecular interactions, particularly protein-protein binding.
• Studying these interactions is crucial for understanding molecular mechanisms.
• Existing methods like fluorescence spectroscopy and surface plasmon resonance often require labeling or immobilization.

Purpose of the Study:

• To introduce and validate Backscattering Interferometry (BSI) as a label-free, immobilization-free technique for biomolecular interaction analysis.
• To demonstrate the application of BSI in studying protein-protein interactions under physiologically relevant conditions.
• To measure the binding affinity between recoverin and G protein-coupled receptor kinase 1 (GRK1) using BSI.

Main Methods:

• Utilized Backscattering Interferometry (BSI) for label-free and immobilization-free interaction analysis.
• Employed a model system involving the neuronal calcium sensor recoverin and its target G protein-coupled receptor kinase 1 (GRK1).
• Performed titration experiments by mixing increasing concentrations of recoverin with a fixed concentration of GRK1 fusion protein.

Main Results:

• BSI enabled sensitive detection of recoverin-GRK1 interactions in small volumes.
• The equilibrium dissociation constants determined by BSI were in excellent agreement with previously reported affinity constants.
• The study provides a detailed protocol for BSI instrumental setup, data acquisition, and evaluation.

Conclusions:

• Backscattering Interferometry (BSI) is a highly sensitive, label-free, and immobilization-free technique suitable for studying biomolecular interactions.
• BSI provides accurate affinity measurements under physiologically relevant conditions.
• This method offers a valuable alternative to existing techniques for analyzing protein-protein interactions, exemplified by the recoverin-GRK1 system.