Single-molecule force spectroscopy data reveal the force required to unfold AIM. (A) The optical tweezers were configured with BioSpy-AIM-A1 fixed between 2 streptavidin-functionalized polystyrene beads. The biotinylated N-terminal AIM was bound directly to the fixed bead, whereas the C-terminal AIM appended with a SpyTag connected to the laser-trapped bead with a DNA handle functionalized with biotin and a SpyCatcher molecule at either end. (B) Plots of the worm-like chain model fitting results of unfolding force vs unfolding extension for each mutant compared with the WTAIM-A1. Force data are presented as mean values ± standard deviation, and extension data are presented as the peak of the Gaussian fit ± the full width at half maximum of Gaussian fit divided by the square root of counts. (C) Plots of Bell-Evans model fitting results of unfolding force vs loading rate for each AIM-A1 mutant compared with the WT AIM-A1. Unfolding force data are presented as the center of the tallest bin of the histogram ± one-half of the bin width. Numerical values of associated fittings are summarized in Table 2.