Quantifying Weak Glycan-Protein Interactions Using a Biolayer Interferometry Competition Assay: Applications to ECL...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Determining the binding affinity (K D) of weak glycan-protein interactions is crucial for understanding biological processes.
• Existing methods often require large amounts of purified reagents or lack precision for weak interactions.

Purpose of the Study:

• To introduce and validate two bio-layer interferometry competition assay formats.
• To accurately determine solution K D values for weak glycan-protein interactions.
• To minimize reagent consumption in binding assays.

Main Methods:

• Utilized bio-layer interferometry (BLI) competition assays.
• Applied the method to Erythrina crista-galli lectin and influenza hemagglutinin (X-31).
• Compared results with isothermal titration calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) data.

Main Results:

• Successfully determined solution K D values for weak glycan-protein interactions.
• Demonstrated good agreement between BLI results and literature values from ITC and NMR.
• Validated the robustness and precision of the developed BLI assay formats.

Conclusions:

• The developed bio-layer interferometry competition assays provide a robust and precise method for determining K D values of weak glycan-protein interactions.
• This approach offers a reagent-efficient alternative for characterizing molecular interactions.
• Accurate K D values are essential for elucidating complex binding phenomena, including monomeric vs. multimeric interactions and affinity vs. avidity.