Mechanically weak and highly dynamic state of mechanosensitive titin Ig domains induced by proline isomerization

Titin, essential for mechano-homeostasis in cardiac and skeletal sarcomere, contains numerous mechanosensitive immunoglobulin-like (Ig) domains in its I-band region. However, how proline isomerization and cysteine-mediated disulfide bond collectively regulate Ig domain dynamics within the physiological force range remains unclear. Here, we use single-molecule force spectroscopy to quantify the proximal Ig1 domain, revealing that proline isomerization leads to two native states-trans and cis states-with distinct mechanical and thermal stabilities. The trans-Ig1 unfolds at forces of ~ 5 pN, which is over 50 pN lower than that of cis-Ig1, and unfolds 1000 times faster under physiological forces. Furthermore, such proline induced dual-state is likely shared feature across majority of I-band Ig domains. Additionally, reduced cis- and trans-Ig1 exhibit catch-slip bond unfolding, while oxidized forms display slip-catch-slip unfolding. This study offers insight into effective modulation of proline isomerization and disulfide bond in regulating mechanosensitive proteins within the physiological force range.