Titin is a Spatially Homogenous Linear Expander

Abstract

Titin, a giant filamentous intrasarcomeric protein, is a serial chain of more than 300 globular (Ig or FN) domains and numerous unique sequences. Although force-dependent unfolding has been extensively investigated in recombinant homopolymeric constructs of titin domains, neither the global kinetics, nor the spatial pattern of mechanically-driven domain unfolding is known within the complexity of the full-length molecule. To follow the global kinetics of domain unfolding, we stretched individual titin molecules isolated from rabbit m. longissimus dorsi using high time- and force-resolution optical tweezers in force- and velocity-clamp modes. When clamped at high forces, the molecule extended in discrete steps via unfolding of its constituent globular domains. In an apparent violation of mechanically-driven activation kinetics, however, neither the global domain unfolding rate, nor the folded-state lifetime distributions of titin were sensitive to force. The contradiction can be reconciled by assuming a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of force increases. To explore whether there is a spatial pattern in this gradient of domain unfolding, we carried out a topographical screening of individual titin molecules stretched to varying degrees with receding meniscus. We found that unfolded domains were distributed homogenously along the entire length of the overstretched titin molecule. The spatially randomized domain stability ensures that titin is a quasi Hookean expander across a wide range of stretch and loading rates, thereby behaving as an apparently linear sensor of the mechanical environment. This could serve as a built-in safety mechanism for protecting the sarcomere against structural disintegration.