Fig. 6: Temperature-dependent α-actinin SR4 unfolding/refolding dynamics.
From: Modulating mechanical stability of heterodimerization between engineered orthogonal helical domains
a Top panel: experimental design of utilizing the split-13-abshear to anchor a protein construct (bio-6SR-b) that contains six repeats of the fourth spectrin-repeat domain to glass surface. The six SR domains are spanned between an avi-biotin tag at N-terminus and the #13-b helix-hairpin at the C-terminus. The bio-6SR-b construct is specifically tethered to the #13-a helix-hairpin coated bottom surface via formation of split-13-abshear complex, and the N-terminus is tethered to the DNA-coated magnetic bead through biotin–streptavidin interaction (Supplementary Note 2). Bottom panel: five representative time traces of the bead height obtained from five independent tethers under a constant force of ~7.9 pN at 23 °C. The six stepwise extension increase events with a step size of ~25 nm in each time trace indicate SR unfolding. Colored lines are 20-points FFT smooth of the raw data (gray). b Representative time traces of the bead height (20-points FFT smoothed) under a constant force of ~5.7 pN at multiple temperatures (23, 27, 29, and 31 °C). The stepwise extension increases and decreases with step sizes of ~18 nm indicate unfolding/refolding of the SR domains. c The resulting probability distributions of the bead height. The numbers of unfolded spectrin-repeat domains at corresponding bead height are indicated. Blue line is the multiple peak Gaussian fitting of the bead height probability distributions. d The bar graph shows the probability of having n unfolded SR domains in the six SR domains obtained from the bead height distribution in c. The error bar indicates the standard error, which are obtained through multiple peak Gaussian fitting of the bead height probability distributions. Blue curve indicates the fitting of the bar graph to the binomial distribution. Source data are provided in Source Data file.
