Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties.

Active muscles generate substantial mechanical forces by the contraction/relaxation cycle, and, to maintain an ordered state, they require molecular structures of extraordinary stability. These forces are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain arrays. Members of the myomesin protein family function as molecular bridges that connect major filament systems in the central M-band of muscle sarcomeres, which is a central locus of passive stress sensing. To unravel the mechanism of molecular elasticity in such filament-connecting proteins, we have determined the overall architecture of the complete C-terminal immunoglobulin domain array of myomesin by X-ray crystallography, electron microscopy, solution X-ray scattering, and atomic force microscopy. Our data reveal a dimeric tail-to-tail filament structure of about 360 Å in length, which is folded into an irregular superhelical coil arrangement of almost identical α-helix/domain modules. The myomesin filament can be stretched to about 2.5-fold its original length by reversible unfolding of these linkers, a mechanism that to our knowledge has not been observed previously. Our data explain how myomesin could act as a highly elastic ribbon to maintain the overall structural organization of the sarcomeric M-band. In general terms, our data demonstrate how repetitive domain modules such as those found in myomesin could generate highly elastic protein structures in highly organized cell systems such as muscle sarcomeres.

Heat shock protein 90's mechanochemical cycle is dominated by thermal fluctuations.

The molecular chaperone and heat shock protein 90 (Hsp90) exists mainly as a homodimer in the cytoplasm. Each monomer has an ATPase in its N-terminal domain and undergoes large conformational changes during Hsp90's mechanochemical cycle. The three-color single-molecule assay and data analysis presented in the following allows one to observe at the same time nucleotide binding and the conformational changes in Hsp90. Surprisingly, and completely unlike the prior investigated systems, nucleotides can bind to the N-terminally open and closed state without strictly forcing the protein into a specific conformation. Both the transitions between the conformational states and the nucleotide binding/unbinding are mainly thermally driven. Furthermore, the two ATP binding sites show negative cooperativity; i.e., nucleotides do not bind independently to the two monomers. We thus reveal a picture of how nucleotide binding and conformational changes are connected in the molecular chaperone Hsp90, which has far-ranging consequences for its function and is distinct from previously investigated motor proteins.

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

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. High-resolution 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.

Direct observation of active protein folding using lock-in force spectroscopy.

Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study, we present a lock-in force-spectroscopy technique that improves resolution of atomic-force microscopy force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from Dictyostelium discoideum filamin (ddFLN4) refolds against forces of approximately 4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide before folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain-length and zero force refolding rate.

Anisotropic deformation response of single protein molecules.

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 half-folded intermediate structure. From potential widths we estimated directional spring constants of the GFP structure and found values ranging from 1 N/m up to 17 N/m. 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 of polyproteins for single-molecule force spectroscopy.

Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.