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. 2022 May 18;2(6):1417–1427. doi: 10.1021/jacsau.2c00121

Figure 3.

Figure 3

Experimental validation of biasing of ddFLN4 and FIVAR unfolding forces by the VHH:mCherry complex and implementation of correction algorithm. (a, b) AFM-SMFS measurements on FIVAR domain with (a) Sdrg:Fg complex (unbiased system) and (b) VHH:mCherry complex (biased system). Experimental AFM setup, representative force trace, and the aligned contour length histogram are shown. The unfolding of FIVAR domain with an ∼31 nm increment, followed by the two-step unfolding of the ddFLN4 FP domain with ∼35 nm increments could be identified from the contour length histogram. (c) Dynamic force spectrum of FIVAR unfolding forces obtained from both (a) unbiased system using SdrG:Fg complex (blue) and (b) biased system using VHH:mCherry complex (red). (d) Dynamic force spectrum of ddFLN4 unfolding forces obtained from both (a) unbiased system using SdrG:Fg complex (blue) and (b) biased system using VHH:mCherry complex (red). The most probable rupture force and loading rates were fit using the Bell–Evans model (dashed lines). Using the fitting approach based on minimizing η residuals, we obtained new energy landscape parameters corresponding to the black dashed line. In the right-hand-side plots of (c) and (d), the black solid line represents the distribution after algorithmic correction.