Figure 1: Mechanical characterization of I27 conformers.

(a) Illustration of 8xI27 (green domains) tethered by the C-terminus to a gold support and N-terminally attached to the tip of the AFM cantilever. (b) Example of force-distance curves recorded upon initial unfolding and after refolding a single 8xI27, accompanied by explanatory illustrations (i–v). 8xI27 (i) is unfolded (ii) when retracting the support (red arrow), rendering the red force-distance curve. Each force peak of the force-distance curve corresponds to the unfolding of a single I27 domain. Thereafter, the tension applied to unfold I27 domains is reduced by re-approaching tip and support indicated by the black arrow (iii), resulting in the black force-distance curve. The collapsed unfolded 8xI27 (iii) is kept relaxed for a given refolding time (1–8 s). During this period some I27 domains may refold (iv). To characterize the adopted conformations, the same protein is once again unfolded (v). The resulting turquoise force-distance curve shows the number of domains refolded (in this example four I27 domains). To avoid unspecific interactions between tip and support, the tip is kept separated from the support by ≈20 nm (close to the contour length of one unfolded I27 domain). The portion of 8xI27 allowed to refold is highlighted by the light yellow background of the force-distance curves. (c) Identifying different I27 conformers in force-distance curves recorded upon initial unfolding (red) and unfolding refolded 8xI27 (turquoise). Force peaks were fitted using the worm-like chain (WLC) model to reveal contour lengths and unfolding forces (black fits). The contour length difference between force peaks is used to characterize I27 conformers. Folded conformers (white squares) are found to have contour lengths separating individual force peaks between 25 and 31 nm and unfolding forces (F) between 50 and 300 pN. Force features that do not meet criteria for properly folded I27 domains originate from misfolded I27 conformers (grey squares). The absence of force features preceding the first force peak reflect I27 domains that did not refold and therefore indicate unfolded conformations (black squares). The bar represents the percentage of I27 conformers detected by the refolding force-distance curve (turquoise).