A Force Spectroscopy-Based Protein-Ligand Interaction Assay

Abstract

Binding of small molecules are crucial to the function and folding of many protein machineries inside cell. Thus it is of fundamental importance to measure the binding affinity of small molecule ligands to proteins and reveal the binding mechanism. Here we report a force spectroscopy based single-molecule binding assay that is capable of determining the binding affinity as well as the binding mechanism of ligands to proteins at the single-molecule level. This assay is based on the difference in the mechanical stability of the given protein upon ligand binding. As a proof-of-principle, we use the binding of metal ions, Ni2+, to an engineered metal binding protein, G6-53, as a model system to establish this method. The apo-G6-53 and Ni2+-bound G6-53 exhibit distinct mechanical stability: apo-G6-53 unfolds at around 120 pN while Ni2+-bound G6-53 unfolds at ∼250 pN. Using their characteristic unfolding forces as a reporter, we were able to directly quantify the partitioning of G6-53 between the apo and Ni2+ bound states at different Ni2+ concentration and measure the binding affinity of Ni2+ to G6-53. The distinct unfolding forces of apo and holo forms of G6-53 also allow us to discriminate different species in the process of folding and Ni2+ binding and measure their kinetic evolution. We unfolded G6-53 by force and waited to allow it to fold and bind with Ni2+. We found that the unfolded G6-53 folds to apo form before incorporating Ni2+. The folding rate of G6-53 is independent of Ni2+ concentration, while the binding rate of Ni2+ to apo form of G6-53 is directly proportional to the Ni2+ concentration. Our kinetic data can be fully described using a “folding before binding” model. We anticipate that this novel assay will find unique applications in the study of various protein-ligand interactions.