Fig. 1: Force-dependent unfolding and refolding of Csp measured by magnetic tweezers at constant loading rates.

a Sketch of magnetic tweezers pulling protein construct of 2xI27-Csp-2xI27 with the AviTag at N-terminus attached to a streptavidin-coated paramagnetic bead and SpyTag at C-terminus attached to the glass surface coated with SpyCatcher. Zoomed in figure shows a structure of Csp. b Extension time course of 2xI27-Csp-2xI27 in a force cycle at a loading rate of ±1 pN s⁻¹ from 1.2 to 25 pN, an unfolding step and a refolding step were observed at ~16.6 and ~4.7 pN, respectively. After waiting for 30 s at 1.2 pN, a force was increased at a loading rate of 1 pN s⁻¹ to 80 pN, additional four unfolding steps I27 were observed after unfolding of Csp. Raw data were recorded at 200 Hz (black) and smoothed in a time window of 1 s (red). c Unfolding force distribution at a constant loading rate of 0.4 pN s⁻¹ were obtained from 81 unfolding events. The fitting of unfolding probability density with Eq. (2) gives a \(k_u^0 = 0.005\) s⁻¹ and \(x_u = 0.97\) nm. d Folding force distribution at a constant loading rate of −0.4 pN s⁻¹ were obtained from 66 folding events. The fitting of folding probability density with Eq. (3) gives a \(k_f^0 = 1.3 \times 10^7\) s⁻¹ and \(x_f = 11.1\) nm. e The average unfolding forces were obtained at a loading rate from 0.4 to 16 pN s⁻¹ from five different tethers. Linear fitting with Eq. (4) gives \(k_u^0 = 0.002\) s⁻¹ and \(x_u = 1.42\) nm at loading rates from 0.4 to 4 pN s⁻¹ (black line) and gives \(k_u^0 = 0.03\) s⁻¹ and \(x_u = 0.64\) nm at loading rates from 4 to 16 pN s⁻¹ (black dashed line). The number of unfolding events at different loading rates are from 34 to 120. Error bar shows the standard error of the mean.