Figure 5. Desialylation of AIM-A1 reduces the mechanical stability of the AIM.

(A) Schematic diagram of the optical tweezer experiment. Carboxyl-polystyrene beads were covalently coupled with streptavidin. Streptavidin beads were incubated with the BioTag-1238–1493-10His-SpyTag construct. SpyCatcher was coupled to biotin-DNA handle, and then incubated with streptavidin beads. For pulling experiments, one bead with SpyCatcher-DNA handle was fixed by the micropipette, while the other bead was trapped and controlled by the optical tweezers. (B) Typical force retraction traces of pulling sAIM-A1 and asAIM-A1 at 500 nm/s. Arrows indicate the occurrence of an unfolding event. (C)Plots of unfolding force versus unfolding extension for sAIM-A1 and asAIM-A1 and fits to the worm-like chain model. Histograms of unfolding extension (shown in the inset as an example with Gaussian fit) was used to determine the peak extension. 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 (FWHM) of Gaussian fit divided by the square root of counts. (D) Plots of the most probable unfolding forces, determined from force histograms (shown in inset as an example), versus loading rates. The error bars are the half bin width. The solid lines are the linear fits of the data to the Bell-Evans model.