Extended Data Fig. 4: The (R16)2-(Ig27WT)2 and (Ig27WT)2-(R16)2 specular polyprotein constructs are mechanically equivalent.
From: Structural anisotropy results in mechano-directional transport of proteins across nuclear pores
(A) Schematics of the (R16)2-(Ig27WT)2 polyprotein construct under force. (B) Representative AFM force-extension unfolding trajectory of the (R16)2-(Ig27WT)2 construct. The protein domains unfold following their mechanical hierarchy. The two first unfolding events, fingerprinted by an unfolding force (FU) of ∼35 pN and an increase in contour length (ΔLC) of ∼37 nm, correspond to the unfolding of the two mechanically labile R16 domains. The two subsequent events, hallmarked by FU∼220 pN with ΔLC∼27 nm, correspond to the unfolding of the stiffer Ig27WT. (C) Scatter plot of unfolding force (FU) versus increase in contour length (ΔLC) for all unfolding events detected on the AFM force–extension trajectories for the (R16)2-(Ig27WT)2 construct. Two clusters of events can be clearly identified by their unique FU and ΔLC signature, a first one with FU = 33 ± 12 pN and ΔLC = 37 ± 4 nm corresponding to the unfolding of the R16 domains (magenta), and a second one with FU = 204 ± 20 pN and ΔLC = 27 ± 2 nm corresponding to the Ig27WT domains. Data collected from n = 61 (R16), n = 78 (Ig27WT), unfolding events from 41 individual force-extension unfolding trajectories. (D-F) Schematics, force–extension trace, and FU versus ΔLC scatter plot for the (Ig27WT)2-(R16)2 construct, showing a first cluster of events characterized by FU = 35 ± 12 pN, and ΔLC = 35 ± 3 nm (R16, magenta) and a second one with Fu = 222 ± 25 pN and ΔLC = 28 ± 2 nm (Ig27WT, grey). When pulled in a single-molecule AFM, both specular constructs are mechanically indistinguishable. Data collected from n = 48 (R16), n = 68 (WT), unfolding events from 43 force-extension individual trajectories measured at 400 nm/s.
