Figure 3: A force-independent binding model.

(a) The kinetic model that considers rapid dynamics between the calcium-free apo and the calcium-bound holo conformations. The unfolding of both the apo and holo forms are force-dependent processes and the interconversion between the apo and holo forms depends on the calcium concentration and is force independent. (b) This kinetic model fails to reproduce the unfolding force histograms at various calcium concentrations. Black bars correspond to the experimentally obtained unfolding force histograms and red lines correspond to the numeric fitting results using the kinetic model depicted in a The χ² for the fittings to the histograms from top to bottom are 0.093, 0.096, 0.308 and 0.290, respectively. (c) This kinetic model cannot describe the unfolding forces of G6 at different calcium concentrations. Red circles correspond to the numerically calculated data using a K_d of 14.4 μM, as determined from ITC measurements. Blue circles correspond to the numerically calculated data using a K_d of 0.05 μM that best ‘fits’ the experimental data. However, the slope of the unfolding force versus [Ca²⁺] relationship cannot be fully reproduced. The χ² for numerically calculated data using K_ds of 14.4 and 0.05 μM are 14.0 and 0.33, respectively.