Abstract
The speed of formation and breaking apart of non-covalent complexes is a key determinant of function in molecular biology and pharmacology. Dissociation rates of complexes are conventionally assumed to depend only on the interactions between the molecules forming the complex. Experiments studying non-covalent interactions sometimes show concentration dependent dissociation rates, which are commonly attributed to the formation of a ternary complex of the binding partners and the competitor. Here we challenge this view through experiments that test the role of competitor molecules in solution in the dissociation process. We use single-molecule fluorescence co-localization experiments to confirm a key prediction of our model in which competitor accelerates dissociation of a non-covalently bound molecular complex by occluding the rapid rebinding of binding partners. The results show that an acceleration of ligand dissociation rate with increasing competitor concentration is a natural feature of a molecular competition that can occur in biologically relevant ranges of competitor concentration. In particular, contrary to common assumption the observation of such acceleration does not necessarily indicate formation of a ternary complex of binding partners and competitor. The results also demonstrate that single-molecule co-localization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution.