Fig. 5: Temperature-dependent titin I27 unfolding/refolding dynamics.

a Experimental design of utilizing the split-13-ab^shear helix-heterotetramer to anchor a protein construct (bio-4I27-b) that contains four repeats of titin I27 domains to coverslip surface. The I27 domains are spanned between an avi-biotin tag at N-terminus and the #13-b helix-hairpin at the C-terminus. The bio-4I27-b construct is specifically tethered to the #13-a helix-hairpin coated bottom surface via formation of split-13-ab^shear complex, and the N-terminus is tethered to the DNA-coated magnetic bead through biotin–streptavidin interaction (details are provided in Supplementary Note 2). b Six representative time traces of the bead height obtained from six independent tethers under a constant force of ~ 8 pN at 23 °C. The four stepwise extension increase events with a step size of ~15 nm indicate I27 unfolding. Colored lines are 200-points FFT smooth of the raw data (gray). c Left panel: a representative time trace of the bead height (20-points FFT smoothed) under a constant force of ~4 pN at 37 °C. The stepwise extension increase/decrease with step sizes of ~10 nm indicates unfolding/refolding of the I27 domains. Middle panel: the corresponding probability distributions of the bead height. Blue line is the multiple peak Gaussian fitting of the bead height probability distributions. Right panel: The bar graph shows the probability of having n unfolded I27 domains in the four I27 domains obtained from the bead height distribution in the middle panel. The error bar indicates the standard error, which is 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, from which the probability of the unfolded state of a single I27 domain at this force is determined to be: \(p\left( F \right)\sim 0.27\) at 37 °C. Source data are provided in Source Data file.