Method to Measure the Activation Energy of the Receptor-Ligand Binding by Single-Molecule Force Spectroscopy

Abstract

Single molecule force spectroscopy (SMFS) is routinely used in biophysical research to measure the kinetic parameters of dissociation of biomolecules by affecting the dissociation kinetics with external force. We have noticed that probability to form bond between tethered ligand and immobilized receptor measured by atomic force microscopy (AFM)-based SMFS depends on time the AFM probe spends near the surface. This dependence has been attributed to the tether-constrained kinetics of forming the molecular bond between the ligand and the receptor. We have developed a kinetic model that incorporates the polymeric tether dynamics and permits measurement of the activation energy of association in bimolecular reaction. To test this model we have performed SMFS measurements using biotin tethered to the AFM probe by PEG linkers with molecular weights of 5 and 3.4 kDa and streptavidin immobilized on a flat substrate. Measurements support the developed model and yield activation energy of the binding of 9.5±0.5 kT. The developed approach can be used as a common SMFS tool in characterizing the activation energy of binding in other molecular systems.View Large Image | View Hi-Res Image | Download PowerPoint Slide