Figure 6. Biasing of unfolding force distributions by dual binding mode.
(A) Typical force traces showing iLOV unfolding with final single (green) and double (purple) complex ruptures. The curve terminating in a double peak is offset in the y-direction for clarity. (B) Final complex rupture force distribution for single and double events. Double events are more mechanically stable. (C) iLOV domain unfolding forces for final single (green) and double (red) events at a pulling velocity of 800 nm/s. Histograms (bars), kernel density estimates (lines), and statistical tests are each obtained from the raw data. Maxima for iLOV unfolding lie at 96.0 pN and 102.7 pN for single (N = 172) and double (N = 277) final ruptures, respectively. A two-sample Kolmogorov-Smirnov test showed significant differences in the data distributions (p-value of 0.09%). Since the data were all recorded with a single cantilever and both event types were distributed equally throughout the runtime of the measurement, no systematic biasing is expected. Because of the lower force distribution of final single peaks, the iLOV unfolding force distribution is truncated compared to final double peak force traces, supporting the notion that the binding mode is set prior to mechanical loading of the complex.