Abstract
When exposed to high forces, titin unfolds in discrete steps via unfolding of its globular domains. By contrast, refolding is strongly inhibited by mechanical force and chain extension, which results in a large force hysteresis in stretch-relaxation cycles. Whereas the stretch force curve of titin is populated by sawtooth-shaped transitions, the relaxation force curve is devoid of significant transitions, making it difficult to capture the refolding event. Although titin refolds if incubated in the contracted state, the exact trajectory of the folding process is unclear. To explore the detail of titin's mechanically-driven folding and unfolding, we manipulated single molecules with high-resolution optical tweezers. Whereas titin extended in discrete steps at high constant forces, after quenching the force to low levels the extension fluctuated without resolvable discrete events but with low frequency (second timescale) and high (several 100 nm) peak-to-peak amplitude. In constant-trap-position experiments at very low (<1 pN) average forces fluctuations were observed, suggesting that the domains hop between an extensible unfolded and a compact molten-globule state. Monte-Carlo simulations based on a compact molten-globule intermediate recovered all features of the force-clamp results. Under mild denaturing conditions (0.5 M urea) that favor the molten-globule state, the length and force fluctuations appeared even in constant-velocity experiments, indicating that this intermediate is part of the folding trajectory. Because the transition from the unfolded to the molten-globule state shortens the chain faster than a purely entropic collapse, an additional sarcomeric contractility may arise under stressed conditions when titin's domains become unfolded.
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