Low-force transitions in single titin molecules reflect a memory of contractile history

Abstract

Titin, a giant elastomeric muscle protein has been implicated to function as a sensor of sarcomeric stress and strain but with unresolved mechanisms. To gain insight into titin's mechanosensory function here we manipulated single molecules with high-resolution optical tweezers. Discrete, stepwise transitions, with rates faster than canonical Ig-domain unfolding occurred during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions (PEVK, proximal tandem-Ig, N2A) are likely to be involved. The pattern of transitions is sensitive to the history of contractile events. Monte-Carlo simulations recovered our experimental results and predicted that structural transitions may begin prior to the complete extension of the PEVK domain. High-resolution AFM of titin extended with meniscus forces supported this prediction. Addition of glutamate-rich PEVK-domain fragments competitively inhibited the viscoelastic response in both single titin molecules and muscle fibers, indicating that intra-PEVK-domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends via a complex pattern of history-dependent discrete conformational transitions which, by dynamically exposing ligand-binding sites, may set the stage for the biochemical sensing of the sarcomeric mechanical status.