Single-Molecule Studies on PolySUMO Proteins Reveal Their Mechanical Flexibility

Proteins with β-sandwich and β-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.     

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species.     

Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin

Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and ¹⁰FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a “mechanical clamp”. Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the “mechanical clamp” geometry at the termini. M-crystallin is a Ca²⁺ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)₈, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and ¹⁰FNIII, as well as lacking “mechanical clamp” geometry, can be mechanically resistant. Furthermore, Ca²⁺ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca²⁺ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.     

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, υ^‡. Here we used single molecule force-clamp spectroscopy to directly determine the value of υ^‡ for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ΔG^‡ = 71 ± 5 kJ/mol and an exponential prefactor υ^‡ ∼4 × 10⁹ s⁻¹. Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (∼6 × 10¹² s⁻¹), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (∼10⁷ m⁻¹ s⁻¹). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.     

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions–diameter (D): 40–50 μm, height (H): 100 μm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.     

Characterisation of the SUMO-like domains of Schizosaccharomyces pombe Rad60

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described, Rad60 has a Hus5-independent function.     

Experimental comparison of energy landscape features of ubiquitin family proteins

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant-dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two-state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow-down their folding rates. The denaturant-dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent-accessible surface area, as quantified by the Tanford β _u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding-unfolding rates within a structurally homologous protein family.     

Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.     

Mechanism of E1-E2 Interaction for the Inhibition of Ubl Adenylation

Conjugates of ubiquitin or its homologues to other proteins occur by strictly ordered steps with ordered addition of substrates for each step. High concentrations of E2 were shown to inhibit the formation of E2∼Ubl thioester and Ubl∼target conjugates. We investigated the mechanism of such inhibitory effect of the SUMO E2 and whether the E2 has two binding sites on its E1, one for the inhibitory effect and one for productive SUMOylation. NMR methods in combination with mutagenesis and biochemical assays revealed that Ubc9 binds to two flexible domains of its free E1 simultaneously, suggesting extensive domain movements in the free E1. Further, interaction of free E1 and E2 inhibits SUMO adenylation, and the interfaces responsible for the inhibition were the same as those required for productive transfer of SUMO from E1 to E2. This study indicates a conformational flexibility-dependent mechanism to control the strictly ordered steps in Ubl modifications.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Protrusive and Contractile Forces of Spreading Human Neutrophils

Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil’s morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell’s mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (−20 ± 10 pN/post) than those residing at the geometric perimeter (−106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β₂ integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.     

E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (k_obs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of k_obs on TDG concentration gives k_max = 1.6 min⁻¹ and K_1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (K_d ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.     

Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

SUMO modification of cell surface Kv2.1 potassium channels regulates the activity of rat hippocampal neurons

Voltage-gated Kv2.1 potassium channels are important in the brain for determining activity-dependent excitability. Small ubiquitin-like modifier proteins (SUMOs) regulate function through reversible, enzyme-mediated conjugation to target lysine(s). Here, sumoylation of Kv2.1 in hippocampal neurons is shown to regulate firing by shifting the half-maximal activation voltage (V(1/2)) of channels up to 35 mV. Native SUMO and Kv2.1 are shown to interact within and outside channel clusters at the neuronal surface. Studies of single, heterologously expressed Kv2.1 channels show that only K470 is sumoylated. The channels have four subunits, but no more than two non-adjacent subunits carry SUMO concurrently. SUMO on one site shifts V(1/2) by 15 mV, whereas sumoylation of two sites produces a full response. Thus, the SUMO pathway regulates neuronal excitability via Kv2.1 in a direct and graded manner.     

Continuous Allosteric Regulation of a Viral Packaging Motor by a Sensor that Detects the Density and Conformation of Packaged DNA

We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼70% and then rises sharply to ∼23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.     

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5′ or 3′ ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.     

Inhibitor binding increases the mechanical stability of staphylococcal nuclease

Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 3′,5′-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ∼26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ∼50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis.     

Molecular Basis of the Mechanical Hierarchy in Myomesin Dimers for Sarcomere Integrity

Myomesin is one of the most important structural molecules constructing the M-band in the force-generating unit of striated muscle, and a critical structural maintainer of the sarcomere. Using molecular dynamics simulations, we here dissect the mechanical properties of the structurally known building blocks of myomesin, namely α-helices, immunglobulin (Ig) domains, and the dimer interface at myomesin’s 13th Ig domain, covering the mechanically important C-terminal part of the molecule. We find the interdomain α-helices to be stabilized by the hydrophobic interface formed between the N-terminal half of these helices and adjacent Ig domains, and, interestingly, to show a rapid unfolding and refolding equilibrium especially under low axial forces up to ∼15 pN. These results support and yield atomic details for the notion of recent atomic-force microscopy experiments, namely, that the unique helices inserted between Ig domains in myomesin function as elastomers and force buffers. Our results also explain how the C-terminal dimer of two myomesin molecules is mechanically outperforming the helices and Ig domains in myomesin and elsewhere, explaining former experimental findings. This study provides a fresh view onto how myomesin integrates elastic helices, rigid immunoglobulin domains, and an extraordinarily resistant dimer into a molecular structure, to feature a mechanical hierarchy that represents a firm and yet extensible molecular anchor to guard the stability of the sarcomere.