Molecular Spring Research Articles

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

(Invited) Rotary Evaporation-Based Preparation of Metastable States

In soft crystals, macroscopic, weak stimulus-induced nanostructural changes often contain the dynamics from the metastable state to the stable state, and therefore, it is important to develop the preparation technologies for metastable states. In this study, I’ll introduce novel approaches for preparing the metastable states of chiral aggregates of phthalocyanines or polythiophenes, i.e., constituents of conducting materials, by using a rotary evaporator. Macroscopic mechanical rotation-induced supramolecular c hirality of phthalocyanines: Fluid dynamics, resulting from the macroscopic mechanical rotation of a rotary evaporator or a magnetic stirrer, has been shown to selectively induce one of two enantiomers (mirror-image structures) in certain nanoscale supramolecules. We demonstrate a novel, highly reproducible, rotary evaporation-induced chiral aggregation of achiral phthalocyanines (Pcs), and proposed the mechanisms for preparing nanoscale chiral supramolecules by the use of macroscopic mechanical rotations. Here, the stable chiral thin films based on H-aggregates of MPcs (M = H2 or Pd) were prepared on the bottom of the flask by concentration of the monomeric solution with a rotary evaporator, and the chirality was shown to reproducibly depend on its rotational direction for the first time. Evaporation rate-based selection of supramolecular chirality of polythiophene: Chirality of poly(3-((S)-3,7-dimethyloctyl)thiophene) (PT-1) aggregates was shown to depend on the rate of evaporation of its solution. In this system, the kinetically favoured, P-type aggregate is maintained in the fast evaporation process, while it transforms to the thermodynamically stable, M-type aggregate in the slow evaporation process. By selectively extracting the metastable P-type aggregate, spontaneous transformation to the M-type aggregate (i.e., molecular power springs) was experimentally demonstrated. This concept will be useful for designing soft crystals with metastable states. References M. Kuroha et al., Angew. Chem. Int. Ed., 2019,58, 18454-18459.Hattori, et al., Chem. Commun., 2017, 53, 3066–3069.Hattori, et al., J. Phys. Chem. B, 2019, 123, 2925–2929.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

Fine‐Tuning the Spring‐Like Motion of an Anion‐Based Triple Helicate by Tetraalkylammonium Guests

AbstractSupramolecular springs are a class of molecular devices that may provide implications to the macroscopic spring behavior from the molecular level. Helical structures are suitable molecular springs because the specific twisting of the helical strands can cause spring‐like (extension‐contraction) movement along the axis upon external stimuli. Herein we report an anion‐based triple‐helicate spring, which can undergo reversible contraction–extension motion through introduction and removal of tetraalkylammonium cations, including TMA+ and analogous irregular tetrahedral cations bearing different alkyl chains, while the relative orientation of the two phosphate ions changes to facilitate guest inclusion. Notably, the degree of contraction (shortening of the helical pitch) can be fine‐tuned by varying the shape and size of guest cation. However, with the larger cations (TEA+, TPA+ and TBA+), the meso‐helicate configuration is obtained, which interconverts with the helicate by addition/removal of TMA+ ion.

Fine-Tuning the Spring-Like Motion of an Anion-Based Triple Helicate by Tetraalkylammonium Guests.

Supramolecular springs are a class of molecular devices that may provide implications to the macroscopic spring behavior from the molecular level. Helical structures are suitable molecular springs because the specific twisting of the helical strands can cause spring-like (extension-contraction) movement along the axis upon external stimuli. Herein we report an anion-based triple-helicate spring, which can undergo reversible contraction-extension motion through introduction and removal of tetraalkylammonium cations, including TMA+ and analogous irregular tetrahedral cations bearing different alkyl chains, while the relative orientation of the two phosphate ions changes to facilitate guest inclusion. Notably, the degree of contraction (shortening of the helical pitch) can be fine-tuned by varying the shape and size of guest cation. However, with the larger cations (TEA+ , TPA+ and TBA+ ), the meso-helicate configuration is obtained, which interconverts with the helicate by addition/removal of TMA+ ion.

Nonsteroidal ecdysone receptor agonists use a water channel for binding to the ecdysone receptor complex EcR/USP.

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing.

TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

Highly tough, stretchable and resilient hydrogels strengthened with molecular springs and their application as a wearable, flexible sensor

Numerous mechanically strong synthetic hydrogels have been developed in recent years; however, few of them are both tough and resilient like living tissues such as muscles. The intrinsically contradictory requirements for toughness and resilience make it a big challenge to design a gel with both high toughness and high resilience. To solve the problem here helical peptide chains are introduced into hydrogel networks by crosslinking the gel with peptide crosslinkers. The resulting hydrogel networks have a reduced inhomogeneity because of the low concentration and large size of the peptide crosslinkers. In addition, under stress the helical chains can be stretched to elongated ones and the intramolecular hydrogen bonds stabilizing the helical structures will be broken, providing a novel mechanism for energy dissipation. Therefore, the peptide-crosslinked hydrogels present significantly improved mechanical strength and extensibility. Unlike the previously used mechanisms for energy dissipation, here the intramolecular hydrogen bonds and hence the helical structure reform instantly when unloaded, leading to a small hysteresis loop and high resilience (>94%). The helical peptide chains in the network act like molecule-sized springs, absorbing and storing mechanical energy when deformed but releasing it when the stress is removed. Therefore, high toughness and resilience are achieved simultaneously. Wearable, flexible strain/pressure sensors were successfully fabricated using the gels. Thanks to the high resilience of the gels, the sensors are highly reliable with unprecedentedly stable baseline and highly reproducible signal.

Urinary Titin N-Fragment as a Biomarker of Muscle Atrophy, Intensive Care Unit-Acquired Weakness, and Possible Application for Post-Intensive Care Syndrome.

Titin is a giant protein that functions as a molecular spring in sarcomeres. Titin interconnects the contraction of actin-containing thin filaments and myosin-containing thick filaments. Titin breaks down to form urinary titin N-fragments, which are measurable in urine. Urinary titin N-fragment was originally reported to be a useful biomarker in the diagnosis of muscle dystrophy. Recently, the urinary titin N-fragment has been increasingly gaining attention as a novel biomarker of muscle atrophy and intensive care unit-acquired weakness in critically ill patients, in whom titin loss is a possible pathophysiology. Furthermore, several studies have reported that the urinary titin N-fragment also reflected muscle atrophy and weakness in patients with chronic illnesses. It may be used to predict the risk of post-intensive care syndrome or to monitor patients’ condition after hospital discharge for better nutritional and rehabilitation management. We provide several tips on the use of this promising biomarker in post-intensive care syndrome.

RBM20-Associated Ventricular Arrhythmias in a Patient with Structurally Normal Heart

RBM20 (RNA-binding motif protein 20) is a splicing factor targeting multiple cardiac genes, and its mutations cause cardiomyopathies. Originally, RBM20 mutations were discovered to cause the development of dilated cardiomyopathy by erroneous splicing of the gene TTN (titin). Titin is a giant protein found in a structure of the sarcomere that functions as a molecular spring and provides a passive stiffness to the cardiomyocyte. Later, RBM20 mutations were also described in association with arrhythmogenic right ventricular cardiomyopathy and left ventricular noncompaction cardiomyopathy. Here, we present a clinical case of a rare arrhythmogenic phenotype and no structural cardiac abnormalities associated with a RBM20 genetic variant of uncertain significance.

Crystals springing into action: metal-organic framework CUK-1 as a pressure-driven molecular spring.

Mercury porosimetry and in situ high pressure single crystal X-ray diffraction revealed the wine-rack CUK-1 MOF as a unique crystalline material capable of a fully reversible mechanical pressure-triggered structural contraction. The near-absence of hysteresis upon cycling exhibited by this robust MOF, akin to an ideal molecular spring, is associated with a constant work energy storage capacity of 40 J g−1. Molecular simulations were further deployed to uncover the free-energy landscape behind this unprecedented pressure-responsive phenomenon in the area of compliant hybrid porous materials. This discovery is of utmost importance from the perspective of instant energy storage and delivery.

Mechanical Flexibility of DNA: A Quintessential Tool for DNA Nanotechnology.

The mechanical properties of DNA have enabled it to be a structural and sensory element in many nanotechnology applications. While specific base-pairing interactions and secondary structure formation have been the most widely utilized mechanism in designing DNA nanodevices and biosensors, the intrinsic mechanical rigidity and flexibility are often overlooked. In this article, we will discuss the biochemical and biophysical origin of double-stranded DNA rigidity and how environmental and intrinsic factors such as salt, temperature, sequence, and small molecules influence it. We will then take a critical look at three areas of applications of DNA bending rigidity. First, we will discuss how DNA’s bending rigidity has been utilized to create molecular springs that regulate the activities of biomolecules and cellular processes. Second, we will discuss how the nanomechanical response induced by DNA rigidity has been used to create conformational changes as sensors for molecular force, pH, metal ions, small molecules, and protein interactions. Lastly, we will discuss how DNA’s rigidity enabled its application in creating DNA-based nanostructures from DNA origami to nanomachines.

Stimuli-responsive Molecular Springs Based on Single- and Multi-stranded Helical Structures

Abstract A wide variety of synthetic molecular machines has been designed and synthesized to construct nanometer-scale assemblies whose molecular motions can be precisely controlled by external stimuli. A helical structure is one of the most intriguing structural motifs to realize such molecular machines, because of its unique spring-like shape that enables reversible extension and contraction motions. This short review highlights the recent progress in the synthesis, structures, and functions of synthetic molecular springs based on single- and multi-stranded helical structures.

The first intracellular loop is essential for the catalytic cycle of the human ABCG2 multidrug resistance transporter.

The human multidrug transporter ABCG2 is required for physiological detoxification and mediates anticancer drug resistance. Here, we identify pivotal residues in the first intracellular loop (ICL1), constituting an intrinsic part of the transmission interface. The architecture includes a triple helical bundle formed by the hot spot helix of the nucleotide‐binding domain, the elbow helix, and ICL1. We show here that the highly conserved ICL1 residues G462, Y463, and Y464 are essential for the proper cross talk of the closed nucleotide‐binding domain dimer with the transmembrane domains. Hence, ICL1 acts as a molecular spring, triggering the conformational switch of ABCG2 before substrate extrusion. These data suggest that the ABCG2 transmission interface may offer therapeutic options for the treatment of drug‐resistant malignancies.

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.

Long-Range Ordered Water Correlations between A–T/C–G Nucleotides

Summary The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology.

Modifications of Titin Contribute to the Progression of Cardiomyopathy and Represent a Therapeutic Target for Treatment of Heart Failure

Titin is the largest human protein and an essential component of the cardiac sarcomere. With multiple immunoglobulin(Ig)-like domains that serve as molecular springs, titin contributes significantly to the passive tension, systolic function, and diastolic function of the heart. Mutations leading to early termination of titin are the most common genetic cause of dilated cardiomyopathy. Modifications of titin, which change protein length, and relative stiffness affect resting tension of the ventricle and are associated with acquired forms of heart failure. Transcriptional and post-translational changes that increase titin’s length and extensibility, making the sarcomere longer and softer, are associated with systolic dysfunction and left ventricular dilation. Modifications of titin that decrease its length and extensibility, making the sarcomere shorter and stiffer, are associated with diastolic dysfunction in animal models. There has been significant progress in understanding the mechanisms by which titin is modified. As molecular pathways that modify titin’s mechanical properties are elucidated, they represent therapeutic targets for treatment of both systolic and diastolic dysfunction. In this article, we review titin’s contribution to normal cardiac physiology, the pathophysiology of titin truncation variations leading to dilated cardiomyopathy, and transcriptional and post-translational modifications of titin. Emphasis is on how modification of titin can be utilized as a therapeutic target for treatment of heart failure.

Modeling a Mechanical Molecular Spring Isolator with High-Static-Low-Dynamic-Stiffness Properties

A mechanical molecular spring isolator (MMSI) is proposed for the purpose of isolating the low-frequency vibration of a heavy payload. The MMSI is a passive vibration isolation technique mimicking molecular spring isolator characteristics of high-static-low-dynamic stiffness (HSLDS). An MMSI consists of a piston-cylinder container filled with the liquid and some hydraulic spring accumulators. The piston would support a lump of mass and be subjected to a specific external vibration excitation force. Those accumulators can get intercommunication by the liquid to produce the transformation from high static stiffness to low dynamic stiffness. The stiffness model of the MMSI with several identical accumulators is established based on the hydrostatic law. After that, some parameters that significantly influence the stiffness characteristics are studied. Results show that the stiffness property of this kind of MMSI demonstrates a piecewise linearity of three segments. It applies the averaging method to acquire amplitude-frequency and phase-frequency relationships of the piecewise linear vibration isolation system. An inevitable jump phenomenon may occur when the exciting force reaches the critical value. The vibration isolation performance is evaluated by energy transmissibility. Finally, an experimental prototype was designed to carry out quasi-static and dynamic experiments to verify the stiffness model and the dynamic properties as an HSLDS vibration isolator.

Cross-Bridges and Sarcomeric Non-cross-bridge Structures Contribute to Increased Work in Stretch-Shortening Cycles.

Stretch-shortening cycles (SSCs) refer to the muscle action when an active muscle stretch is immediately followed by active muscle shortening. This combination of eccentric and concentric contractions is the most important type of daily muscle action and plays a significant role in natural locomotion such as walking, running or jumping. SSCs are used in human and animal movements especially when a high movement speed or economy is required. A key feature of SSCs is the increase in muscular force and work during the concentric phase of a SSC by more than 50% compared with concentric muscle actions without prior stretch (SSC-effect). This improved muscle capability is related to various mechanisms, including pre-activation, stretch-reflex responses and elastic recoil from serial elastic tissues. Moreover, it is assumed that a significant contribution to enhanced muscle capability lies in the sarcomeres itself. Thus, we investigated the force output and work produced by single skinned fibers of rat soleus muscles during and after ramp contractions at a constant velocity. Shortening, lengthening, and SSCs were performed under physiological boundary conditions with 85% of the maximum shortening velocity and stretch-shortening magnitudes of 18% of the optimum muscle length. The different contributions of cross-bridge (XB) and non-cross-bridge (non-XB) structures to the total muscle force were identified by using Blebbistatin. The experiments revealed three main results: (i) partial detachment of XBs during the eccentric phase of a SSC, (ii) significantly enhanced forces and mechanical work during the concentric phase of SSCs compared with shortening contractions with and without XB-inhibition, and (iii) no residual force depression after SSCs. The results obtained by administering Blebbistatin propose a titin-actin interaction that depends on XB-binding or active XB-based force production. The findings of this study further suggest that enhanced forces generated during the active lengthening phase of SSCs persist during the subsequent shortening phase, thereby contributing to enhanced work. Accordingly, our data support the hypothesis that sarcomeric mechanisms related to residual force enhancement also contribute to the SSC-effect. The preload of the titin molecule, acting as molecular spring, might be part of that mechanism by increasing the mechanical efficiency of work during physiological SSCs.

On the Design of a Type of Mechanical Molecular Spring Isolator

The mechanical molecular spring isolator (MMSI) presents the characteristics of high-static-low-dynamic-stiffness to isolate the low-frequency vibration of a heavy payload. An MMSI consists of a piston-cylinder container full of the liquid and some hydraulic spring accumulators. This paper focuses on a particular type of the MMSI that all hydraulic spring accumulators applying the conventional coil spring are identical. In the beginning, the MMSI stiffness model is modelled and the influences of critical parameters on the stiffness property are studied. The MMSI presents a piecewise linear stiffness property. The averaging method is applied to determine the primary resonance response, and the energy transmissibility is adopted to estimate the vibration isolation performance. A jump phenomenon is inevitable to be occurred around the resonance region. The jump avoidance is a critical design part of the MMSI isolation system. On the other hand, based on the stiffness model, it reveals that any stiffness of the coil spring or any size of the accumulator in the MMSI will be able to achieve the same desired stiffness property. The most significant difference between all solutions depends on the coil spring lateral stability. The coil spring lateral stability magnitude is defined as the ratio of the deflection to the buckling deflection to evaluate the quality of each solution. Then, the fundamental design criterion on the MMSI based on jump avoidance and the coil spring lateral stability is established.