Single-molecule methods have given experimental access to the mechanical properties of single protein molecules. So far, access has been limited to mostly one spatial direction of force application. Here, we report single-molecule experiments that explore the mechanical properties of a folded protein structure in precisely controlled directions by applying force to selected amino acid pairs. We investigated the deformation response of GFP in five selected directions. We found fracture forces widely varying from 100 pN up to 600 pN. We show that straining the GFP structure in one of the five directions induces partial fracture of the protein into a halffolded intermediate structure. From potential widths we estimated directional spring constants of the GFP structure and found values ranging from 1 N͞m up to 17 N͞m. Our results show that classical continuum mechanics and simple mechanistic models fail to describe the complex mechanics of the GFP protein structure and offer insights into the mechanical design of protein materials.cysteine engineering ͉ GFP ͉ protein mechanics ͉ protein stability ͉ single-molecule force spectroscopy M any processes in living systems, such as cell division, locomotion, and enzyme activity, depend critically on single protein molecule properties like mechanical rigidity or conformational changes (1, 2). The invention of single-molecule manipulation techniques has given experimental access to the mechanical properties of soluble protein molecules (3-8) and membrane proteins (9-12). Current recombinant protein expression naturally links individual protein modules by their N and C termini. Experimental access to the deformation response of proteins has, thus, so far been limited almost exclusively to one direction of force application: the N-to C-terminal linkage direction of polyproteins. Previous experiments with two proteins that naturally exhibit a non-N-to C-terminal linkage have indicated that the mechanics of protein structures depend on loading geometry (13,14). Theoretical studies have predicted that the deformation response of proteins may vary largely, even on the single-residue level, and that protein structures may contain ''soft'' and ''stiff'' regions (15). In this study, we employ cysteine engineering (16) to gain precise control over the points of force application to a single protein structure. Fig. 1 illustrates the quality of information that controlled force application can supply about the mechanics of a single protein. Fracture or unfolding of a protein structure proceeds on a high-dimensional energy surface determined by multiple weak interactions between amino acid residues (17). Such an energy landscape is schematically shown in Fig. 1. Conventional N-to C-terminal linkage mechanically probes only a single direction (e.g., pathway I in Fig. 1). In turn, complete control of linkage topology makes previously hidden regions of the energy landscape accessible. Directions in which the protein appears soft and fractures easily will be characterized by low-and shallowe...

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Mechanical stability of bonds and protein interactions has recently become accessible through single molecule mechanical experiments. So far, mechanical information about molecular bond mechanics has been largely limited to a single direction of force application. However, mechanical force acts as a vector in space and hence mechanical stability should depend on the direction of force application. In skeletal muscle, the giant protein titin is anchored in the Z-disk by telethonin. Much of the structural integrity of the Z-disk hinges upon the titin-telethonin bond. In this paper we show that the complex between the muscle proteins titin and telethonin forms a highly directed molecular bond. It is designed to resist ultra-high forces if they are applied in the direction along which it is loaded under physiological conditions, while it breaks easily along other directions. Highly directed molecular bonds match in an ideal way the requirements of tissues subject to mechanical stress.atomic force microscopy ͉ force spectroscopy ͉ protein engineering ͉ protein folding

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...