a Schematic representation of HAP of Cdh23 using AFM where the C-terminus of Cdh23 is covalently attached to glass coverslip using sortagging. The N-terminal of Cdh23 is recombinantly modified with biotin and pulled by streptavidin-coated cantilevers. b Scheme of PAP configuration depicting the partner-assisted pulling of Cdh23 EC1-27 with Pcdh15 EC1-2 (red). c and e Representative force-extension curves of Cdh23 in HAP (black) at 2000 nm s−1 and 3000 nm s−1 pulling speeds, respectively. Both curves depict an initial long stretch followed by sawtooth patterns. d and f Representative force-extension features of Cdh23 in PAP (red) at 2000 nm s−1 and 3000 nm s−1 pulling speeds respectively. Both curves exhibit multiple unfolding peaks at the initial stretches, followed by sawtooth patterns of unfolding. The dotted green lines in (c–f) are the WLC model fit. g and h Distributions of ΔLc of Cdh23-stretching in HAP (black line) (n = 838 unfolding events) and PAP (red) (n = 946 unfolding events), respectively. The data are combined for all loading rates. Solid lines represent the Gaussian fits exhibiting four unfolding peaks. i Distributions of initial long and short stretches of Cdh23 obtained in HAP (black) and PAP (red), respectively. The data are plotted for all the force curves obtained at different pulling velocities. Gaussian fittings of the histograms (solid black line) exhibit a mean length gain of 75.0 ± 2.2 nm in HAP and 6.5 ± 0.3 nm in PAP. Errors mentioned are the standard error of fitting. j Probability distributions of four different stretches of Cdh23 in HAP (black) and PAP (red).
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