Fast-folding α-helices as reversible strain absorbers in the muscle protein myomesin

Abstract

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...

Towards design principles for determining the mechanical stability of proteins

“…Proteins are biological nanomachines that utilise mechanical forces in a wide range of cellular processes [1][2][3][4] . These important processes range from the translocation of proteins/DNA across membranes 5,6 , the degradation of proteins by molecular 30 chaperone proteins 7 , the mechanical resilience of proteins within a molecular scaffold [8][9][10][11] and the conversion of mechanical signals into electrochemical signals 12, 13 ( Figure 1).…”

Catch bond-like kinetics of helix cracking: Network analysis by molecular dynamics and Milestoning

“…This non-monotonic behavior is reminiscent of the "catch-bond" phenomenon observed in some inter-molecular structures in which intermediate loads induce unfolding more slowly than either low or high loads. [16][17][18][19][20][21][22][23][24][25][26] The MFPT calculated at 10 pN was longer than that calculated at both 0 pN and 100 pN. Pathway calculations indicated that this non-monotonic behavior appeared to be related to the formation of non-native (i.e., non α-helical) structures, namely π helices, more prominently at this intermediate load level.…”

“…Load magnitudes tested included 0, 5,10,15,20,25,30,40,70, and 100 pN. The load direction, fixed throughout the simulations, was set in the [0 0 1] direction corresponding to the helix's nominal axis as defined by the vector between the center of mass of the load end groups.…”

“…Experimental determinations of helical protein strength via atomic force microscopy have placed the unfolding force in the 20-40 pN range. 25,26 The use in simulations of significantly higher loading rates than in the experiment was attributed to time-scale limits of straightforward simulations. Simulations of unfolding under load are restricted to microseconds at most, far shorter than typical experimental times of milliseconds and longer.…”

“…A recent look at unfolding time versus load for a simple analytical energy landscape with a single energy barrier 25 illustrated that a "rollover" in lifetime can appear when a degree of freedom other than the molecular extension (i.e., load direction) is essential for the unfolding kinetics. This study demonstrated that this "rollover" can occur for a single unfolding pathway, and therefore may not require multiple distinct pathways such as a "fast" and "slow" pathway whose relative traffic switch due to load magnitude.…”