Can kinetic and thermodynamic parameters of protein-ligand dissociation be extracted from AFM pulling data?

Atomic Force Microscopy (AFM) is a promising tool to measure dissociation rates and binding distances in the single-molecule regime. However, interpretation of the obtained rupture data can be challenging. The unbinding process between two complex molecules is often characterized by a spectrum of barrier heights that cannot be parametrized by a single barrier height. In standard analysis, different barriers can be discerned only by measuring over at least 4-5 orders of magnitude of the pulling force rate, which can be difficult to achieve with AFM. Here, we used fits of the full rupture force histograms to address this complexity. We found that multiple unbinding paths can be discerned from measurements at just a few force rates using this approach. We also show how multiple rupture events - situations where multiple bonds breaking simultaneously appear as single rupture events - can be addressed in the analysis.