Distortion of Protein Receptor Decreases the Lifetime of Receptor-ligand Bond

Abstract

Ligand competition assay is often used in single-molecule force spectroscopy (SMFS) to test the specificity of binding. We have noticed that in the SMFS measurements that utilize biotin tethered to the tip of an atomic force microscope and streptavidin bound to the surface, addition of ∼1mM of free biotin in solution does not completely eliminate binding events as detected by SMFS. We hypothesize that the compressive force applied to the streptavidin-biotin complex on the substrate during the measurements shortens the bond lifetime. We have tested this hypothesis by performing a series of measurements with different maximum compressive force applied to the surface. These measurements indicate that the compressive force affects the number of interactions measured in the presence of free biotin. The measured dependence agrees with the model that takes into account the increase of the tip-surface contact area with an increase of the maximum applied force. These results indicate that for SMFS to be used as a competition assays, shortening of a lifetime of the receptor-ligand bond by compressive force should be considered.