Fast-folding alpha-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. 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.

Fig. 1.

Force spectroscopy on My12 homooctamers. (A) Schematic representation of a My12 homooctamer in the AFM experimental setup (not to scale). The blue squares and the black bars represent the My12 Ig domains and their linker helices, respectively (compare to Inset with cartoon representation of the My12-My13 structure) (B) Typical force-extension trace of (My12)₈ unfolding. The circle marks a single unfolding event; black traces represent worm-like chain fits providing the contour length increases ΔL of a single unfolding. The arrows indicate the force plateau. (C) Scatter plot of unfolding forces and corresponding contour length increases with respective distributions (red). The black histogram gives the plateau force distribution and the dashed line its mean value. (D) (My12)₈ trace with coincidental double Ig-domain unfolding. The force plateau (arrows) can be observed before and after this event. (E) Force-extension trace containing stretch (red) and relax (blue) cycles to test the reversibility of the plateau. In the final stretching cycle unfoldings of the Ig domains can be observed.

Fig. 2.

Force-probe molecular dynamics simulation of My12-My13 dimer unfolding. The red force-extension trace clearly exhibits a plateau. Corresponding structural snapshots at 2, 5, 10, and 16 nm are given below in cartoon representation and show that the plateau corresponds to α-helix unfolding. The individual conformational state of both helices is mapped for each residue against extension (Top).

Fig. 3.

Fast and reversible unfolding transitions of the My12 α-helical linker. (A) Schematics of the construct. The cysteine mutation is colored yellow. (B) Typical force-extension trace of My12^G1548C(GB1)₂ dimer unfolding. The light trace is the original data, the dark trace is filtered. In the red part the pulling velocity was 5 nm/s, in the orange part 100 nm/s. The first four peaks correspond to GB1 unfolding, the last one to detachment of the sample. Black lines are worm-like chain fits. The arrow marks the region of fast transitions. (C) Zoom into transitions region. In a representation where the data are plotted as force vs. time rapid transitions between folded and unfolded states are clearly visible. Blue lines follow worm-like chain elasticity. (D) (Upper) The transitions region plotted as (filtered) contour length against time. Blue dashed lines correspond to worm-like chain traces in C. The corresponding contour length histogram (Right) is well described by three Gaussians (blue lines). (Lower) Three zooms into the indicated regions of the trace above. (E) Force-dependent folding (open circles) and unfolding (filled circles) rates as obtained from the analysis of the transition traces (compare to SI Materials and Methods). The gray areas illustrate the variability of meaningful extrapolations to zero-force following the model of Schlierf et al. (32, 33), the dashed lines are linear fits according to the Bell model (30, 31).

Fig. 4.

Forced dissociation of linked My13 dimer domains. (A) Typical force-extension trace of (Ubi)₃My11-My13 linked dimers. A schematic representation of the construct is given on top. Each monomer consists of three ubiquitins (orange) and the myomesin domains My11-My13 (purple, blue, and cyan). The two monomers are linked via an unstructured amino acid sequence (red) with C-terminal cysteines (yellow) that form a covalent disulfide bond. Dimer dissociation (red circle) is identified by a shorter contour length increase than for domain unfolding. (B) Zoom into part of (A) where dimer dissociation occurs. Insets show structure of the My13 domains (cyan) with linker (red) and bonded cysteines (yellow) in the dimeric (Left) and dissociated state (Right). The dissociation force distribution to the right (red circles) is well reproduced by a Monte Carlo simulation (black trace).