Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation

Area of Science:

• Biophysics
• Cell Biology
• Biochemistry

Background:

• Receptor-ligand interactions are fundamental to cellular adhesion.
• Traditional methods for measuring dissociation constants (Kd) are unsuitable for surface-bound molecules in cell adhesion.
• A new approach is needed to quantify 2D binding characteristics under force.

Purpose of the Study:

• To develop and validate a novel method for measuring 2D binding characteristics of surface-attached receptors and ligands.
• To quantify the relationship between bond force and binding affinity in cellular adhesion.
• To apply the method to E-selectin-mediated tumor cell adhesion.

Main Methods:

• A novel centrifugation assay was developed to quantify cell adhesion.
• A stochastic model was formulated, solved, and tested to couple bond force with binding affinity.
• The method was applied to analyze tumor cell adherence to recombinant E-selectin.

Main Results:

• The novel method successfully quantified 2D binding characteristics of surface-attached molecules.
• The estimated zero-force 2D dissociation constant (Kd) for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2).
• The bond interaction range was found to be subangstrom, with fewer than 5 bonds mediating adhesion.

Conclusions:

• The developed method provides a reliable way to measure 2D binding characteristics and forces in cell adhesion.
• The findings offer new insights into the molecular mechanisms of E-selectin-mediated cell adhesion.
• This approach is valuable for studying receptor-ligand interactions in various biological contexts.