Fig. 4
Dual-marker orthogonal fingerprinting overcomes confounding protein dimerization in concurrent atomic force spectroscopy. a Mechanical unfolding of (C3-SUMO1)4 and (C3-L)4 are measured in separate traditional experiments. Individual traces are classified according to their number of 16 and 20 nm unfolding events, which mark the mechanical unfolding of Protein L and SUMO1, respectively. The plot shows the frequency of the traces that have different combination of unfolding events, as indicated by the size of the dots. Traces coming from mechanical unfolding of (C3-SUMO1)4 are assigned when n(16 nm) = 0 and n(20 nm) > 1 (green rectangle), whereas (C3-L)4 traces are identified by n(16 nm) > 1 and n(20 nm) = 0. Number of traces analyzed: (C3-L)4, 163; (C3-SUMO1)4, 525. b Mechanical unfolding of (C3-SUMO1)4 and (C3-L)4, as measured concurrently in orthogonal fingerprinting (OFP) experiments (temporal sequence is represented by Δt; the protein being pulled at each time is highlighted). The plot shows the frequency of the traces that have different combination of unfolding events, as indicated by the size of the dots (298 traces). The gating strategy defined in a allows the classification of the traces as originating from (C3-SUMO1)4 (green rectangle) or (C3-L)4 (black rectangle). c Experimental cumulative unfolding probability distribution of the C3 domain in the context of (C3-L)4 (6 experiments, 177 events, black; data are also presented in Fig. 3c) and (C3-SUMO1)4 (8 experiments, 742 events, green), as measured in traditional experiments. d Experimental cumulative unfolding probability distribution of the C3 domain in the context of (C3-L)4 (873 events, black) and (C3-SUMO1)4 (1043 events, green), resulting from unfolding data obtained in 14 independent OFP experiments. Relative standard deviation (RSD) values in the insets in c, d are estimated using Monte Carlo simulations that consider extreme values of calibration uncertainty (CU). The pulling rate in all experiments was 40 pN s−1
