In the current view of muscle contraction, the power stroke of a myosin motor is the sole source of mechanical energy driving the contraction of muscle. These models exclude titin, the largest protein in the human body, which sets the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils and that the physiological forces on titin fall between 6-8 pN. We utilize magnetic tweezers together with HaloTag anchoring to record the extension of titin Ig domains I10 and I91 for several hours at nanometer resolution in passive force-clamp mode. Titin Ig domains that had been previously unfolded undergo spontaneous stepwise folding at forces below 10 pN. We show that under a pulling force of 8 pN, previously unfolded I10 or I91 domains undergo a stepwise contraction of 13 nm, as they move from the collapsed to the molten globule state. Thus, folding of a single Ig domain at the physiological force of 8 pN, generates 105 zJ of contractile energy, which is larger than the mechanical energy delivered by the power stroke of a single myosin motor (∼40 zJ). From published measurements of passive muscle elasticity, we calculate that both “soft” and “stiff” muscles operate over a range of forces that straddle the folding probability of Ig domains as shown here. We propose that the stretching of an inactivated muscle recruits Ig domains to the unfolded state; activation of the myosin motors reduces the force on titin to harness the energy of Ig domain folding. From this perspective, both the myosin motors and titin operate as an inextricable molecular system for the storage and delivery of mechanical energy.
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