The highly oriented filamentous protein network of muscle constantly experiences significant mechanical load during muscle operation. The dimeric protein myomesin has been identified as an important M-band component supporting the mechanical integrity of the entire sarcomere. Recent structural studies have revealed a long α-helical linker between the C-terminal immunoglobulin (Ig) domains My12 and My13 of myomesin. In this paper, we have used single-molecule force spectroscopy in combination with molecular dynamics simulations to characterize the mechanics of the myomesin dimer comprising immunoglobulin domains My12-My13. We find that at forces of approximately 30 pN the α-helical linker reversibly elongates allowing the molecule to extend by more than the folded extension of a full domain. Highresolution measurements directly reveal the equilibrium folding/ unfolding kinetics of the individual helix. We show that α-helix unfolding mechanically protects the molecule homodimerization from dissociation at physiologically relevant forces. As fast and reversible molecular springs the myomesin α-helical linkers are an essential component for the structural integrity of the M band.atomic force microscopy | protein folding F ilamentous modular proteins play a key role in the force-bearing structures of the sarcomere (1, 2). The most prominent example is the giant muscle protein titin. For titin, a detailed mechanical hierarchy ranging from entropic stretching of unstructured segments over mechanical kinase activation to unfolding of individual domains has been described (3, 4). Whereas in the sarcomeric I band titin provides the muscle with its passive tension (5), the mechanical properties of the M-band section are less well understood. Here, the 185 kDa protein myomesin (6) as well as other filamentous proteins form a large network constituting, together with metabolic enzymes and kinase domains, a well-organized compartment that has both structural and metabolic properties (7). Myomesin comprises 13 domains, with the first one (My1) being unique and the others (My2-My13) either of the immunoglobulin (Ig) or fibronectin type III fold (8). It is part of a complex network that involves interactions with myosin, titin, obscurin, and obscurin-like 1 (9, 10). Through its N-terminal myosin binding domain (My1) and the ability to form antiparallel homodimers via an interface residing in its C-terminal domain (My13) (11), myomesin acts as a cross-linker of myosin in the M band and its presence is crucial for proper M-band organization (12). Ehler et al. have shown that, together with the C-terminal part of titin, myomesin is a requirement for the integration of myosin into the sarcomere; they further suggest that myomesin in the M band, α-actinin in the Z disk, and titin in between form the basic stabilizing structure of the sarcomere (13). This implicates that myomesin is one of the key factors in maintaining the structural integrity of the M band under load.During normal muscle operation, the M band and consequently myomes...