Fig. 4: Mechanical work generated by titin folding.

a We follow a three-pulse force protocol. During the fingerprint pulse at high forces (1), Ig domains are recruited to the unfolded state. These domains are allowed to refold during the long refolding pulse at low force (2). To estimate folding fractions, we measure the number of domains that unfold in the final probe pulse (3), and compare with the initial number of unfolding domains in the fingerprint pulse. Arrowheads point to unfolding events in the probe pulse. The trace was obtained using gastrocnemius HaloTag-TEV titin. b Folding probability of the I-band of HaloTag-TEV titin isolated from gastrocnemius muscle (blue symbols). Solid line shows a sigmoidal fit to the data. The folding probability is less cooperative that the one obtained with engineered titin polyproteins (in gray, data from Rivas-Pardo et al.¹⁵), while the P_F0.5 remains very similar. Data are presented as mean values ± SD, n = 120 total measurements. Source data are provided as a Source Data file. c Force dependency of the work delivered by titin ___domain folding, which is obtained by multiplying step sizes by force (blue symbols). The solid line considers the step sizes obtained by the worm-like chain model. Considering the probability of folding (blue dashed line, data from panel b), we calculate the expected work delivered by titin folding (black dashed line, filled area), which peaks at 6.3 pN producing 41.5 zJ.