By combining springs with motors and chemical switches biological systems can achieve great scales of mechanical power amplification, for example when jumping or launching a projectile. However, the molecular mechanisms of how this is achieved remain unknown. The giant muscle protein titin, composed of hundreds of tandem Ig domains, is a complex elastic protein whose role in muscle function is still poorly understood. A crucial recent discovery that we have made is that titin domains do a surprisingly large amount of mechanical work when they fold against an opposing mechanical force. We have shown that the amount of mechanical work done by a folding titin Ig domain can be 2-3 times larger (～120 zJ) than that of the chemically powered motor myosin II; ～38 zJ. Titin molecules store mechanical energy by unfolding and extending under force. Elastic energy is stored this way by stretching caused by gravitational pulling during locomotion, inertia, chemical modifications, and ATP powered sources to name a few. Titin unfolding occurs at varying rates over a very wide range of forces above >8 pN. By contrast, most of the stored mechanical energy is delivered back only over a small range of forces where the folding probability increases from 0 to 1 (< 6 pN) and the folding protein does large amounts of mechanical work. Thus, protein folding/unfolding is likely to operate as a mechanical battery where different types of energy sources are stored, and then converted back into contractile power.
Given that titin is now known to be the third filament of muscle, determining if protein folding can deliver work quickly enough to match the power output of the myosin motors, is a central question to be answered. The mechanical power output of protein folding is a novel concept and thus has never been studied before.
Cryptic cysteine residues are common in the elastic I band region of titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding Ig domain.
Here we use magnetic tweezers force spectroscopy to study the folding dynamics of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time. We show that the midpoint folding probability of the parent Ig domain reversibly shifts up from 4.0 pN to 12.8 pN upon oxidation. In this force range, the folding contraction dominates the elastic recoil of the protein, delivering stepwise mechanical work which depends on the oxidation state in an all-or-none manner. For example, the output power of a folding contraction at 6 pN goes from 0 zW to 6,000 zW upon introduction of the disulfide bond. This large amount of power is delivered by folding at forces where single molecular motors are typically stalled. From our observations we predict that during muscle contraction, activation of myosin II motors by Ca⁺⁺ leads to a drop in the force experienced by titin, triggering delivery of mechanical power by titin folding. Thus, it seems inevitable now that the three filaments of the muscle sarcomere act in concert to both store and deliver mechanical power, revolutionizing our understanding of the molecular mechanisms of muscle contraction.