Abstract
Titin is a filamentous protein that spans the half sarcomere and functions as a molecular spring, a sarcomeric template, and possibly as a mechanosensor. The force versus extension curve of titin, recorded in constant-velocity experiments, is characterized by entropic-chain behavior onto which discrete contour-length steps caused by domain unfolding are superimposed. The details of the complex mechanical behavior of titin remain hidden, however.To investigate the detail in titin's force-driven folding and unfolding behavior, here we stretched single molecules of skeletal-muscle titin with force-feedback optical tweezers. Titin was extended and relaxed in subsequent stages of constant high (>30 pN) and low (<5 pN) forces, respectively. At high forces titin extended in ∼28-nm steps distributed exponentially as a function of time. The 28-nm steps are assigned to all-or-none unfolding events of titin's globular domains. Significant domain unfolding occurred at physiologically relevant time- (<1 s) and force-scales (<40 pN), suggesting that in vivo titin extensibility may involve repeated domain unfolding and refolding. At low forces, titin refolded in a highly force-dependent manner. Surprisingly, at forces below 1 pN, no significant refolding occurred on a time scale of 10 minutes. By contrast, refolding was strongly facilitated if the force was clamped at moderately increased levels (2-5 pN). Conceivably, mechanical force acts as a chaperone by limiting access to futile parts of the highly complex folding landscape of the full-length titin molecule.
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