Force Spectroscopy and Dissociation Kinetics of Single Molecules Under an Applied Force

The loading rate dependence of the unbinding force of a single ligand/receptor complex is determined by the rate of dissociation (off-rate) in function of applied force. Because the energy barrier for dissociation is lowered proportional to an applied force the off-rate increases exponentially with the force. This leads to a linear dependence of the unbinding force on the logarithm of the loading rate and allows to determine the thermal off-rate by extrapolating to zero force from measurements at finite forces, where the off-rates are much faster [1-3]. However, at larger applied forces the dissociation kinetics might not be determined by the energy barrier that determines the thermal off-rate. To understand what determines the dissociation kinetics in such a case we discussed a simple model which consists of two barriers along the ligand/receptor separation path, i.e. the dissociation proceeds via an intermediate state. It turns out that at small applied force the off-rate is in fact determined by the thermal barrier (independent of the location of the intermediate state). At larger forces however, a cross-over to a regime occurs where the off-rate is either determined by the transition from the ground to the intermediate state or from the intermediate to the dissociated state (depending on the location of the intermediate state). A change of the rate dominating dissociation step with increasing force is possible in this regime. In consequence, an energy landscape with two barriers can show up to three force intervals with a different (exponential) dependence of the off-rate on the force. The unbinding force of the avidin-biotin complex shows three intervals where its loading rate dependence is different [2]. This can be attributed to the presence of at least one intermediate state along the mechanical separation path.