Theory for measuring bivalent surface binding kinetics using total internal reflection with fluorescence...

Area of Science:

• Biophysics
• Surface Science
• Biochemistry

Background:

• Total internal reflection with fluorescence photobleaching recovery (TIR-FPR) is a technique for studying surface reaction kinetics.
• Previous work established theory for monovalent ligand-surface interactions.
• Understanding bivalent binding is crucial for complex biological systems.

Purpose of the Study:

• To extend TIR-FPR theory to accommodate sequential bivalent surface attachment mechanisms.
• To develop methods for extracting kinetic rates from experimental data.
• To provide a theoretical framework for analyzing antibody-membrane interactions.

Main Methods:

• Theoretical extension of TIR-FPR to sequential bivalent binding models.
• Development of algorithms to derive intrinsic surface association and dissociation rate constants.
• Application of the theory to simulated and experimental TIR-FPR data.

Main Results:

• The study presents a validated theoretical model for TIR-FPR analysis of sequential bivalent surface binding.
• Methods are described for accurately determining kinetic rates (kon, koff) from photobleaching recovery curves.
• The enhanced theory is shown to be applicable to complex ligand-surface interactions.

Conclusions:

• The extended TIR-FPR theory provides a robust framework for studying bivalent surface binding kinetics.
• This advancement facilitates the analysis of interactions involving multivalent proteins like antibodies.
• The findings are significant for research on supported membranes and biomolecular surface interactions.