Fig. 3: Weak mechanical stability of the human α-actinin 1 homo-dimer under unzip-stretching force geometry.

a The illustration of the single-molecule construct and experimental design for quantifying the mechanical stability of the dimer under unzip-stretching force geometry. The biotinylated AviTag was placed at the N-terminus of the first rod, while the SpyTag was placed at the C-terminus of the second rod, resulting in unzip-force geometry when force was applied via these two tags. The inset is the theoretically predicated force-dependent rupture stepsizes of the dimer assuming the releasing of the looped linker under unzip-stretching geometry, while all SR domains remain folded. b The representative force–bead height curve of α-actinin 1 rod homo-dimer under unzip-stretching force geometry during force-increase scan (5 pN s⁻¹, red line) and force-decrease scan (−5 pN s⁻¹ to ~ 7 pN, and then −0.1 pN s⁻¹ to ~1 pN, blue line). The rupture event is noted by a red arrow, the unfolding events of the eight domains are noted by eight gray arrows, the re-dimerization event is noted by a blue arrow. The refolding events region is indicated by a dashed elliptic. c The colored lines are representative time–bead height curves of the dimer at three target force examples (bottom to top: 4.2 pN, 5.2 pN, and 6.3 pN, respectively). d The time evolution of the rupture probability under three example forces, from which the characteristic lifetime of the corresponding force was obtained by exponential fitting (\(p(t)=1-\exp (-\frac{t}{\tau })\)). e The force-dependent lifetime of the human α-actinin 1 homo-dimer under unzip-stretching force geometry. The dwell times of the dimer at each force were plotted in gray hollow symbols, and the characteristic lifetime of each force was plotted in solid symbols. The blue line is Bell model fitting curve of the blue data sets, and gives k₀ and Δ indicated on the panel. Source data are provided as a Source Data file.