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Small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy were used to investigate structural peculiarities of two types of amyloid aggregates of smooth muscle titin, which differed in their morphology and ability to disaggregate, and differently bound thioflavin T dye. SAXS showed that the structure/shape of the two titin aggregate types was close to a flat shape. FTIR spectroscopy revealed no differences in the secondary structure of the two types. These data suggest that both types of "flat-shape" titin aggregates are identical in their secondary structure and, as shown previously, have a quaternary cross-β structure. An assumption was made that the most stable supramolecular complexes of a cross-β structure, which do not differ in their secondary structure, formed first during the aggregation of smooth muscle titin. Then, depending on ambient conditions, these supramolecular structures could form titin aggregates of different morphology and properties., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal β-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2., (© 2024. The Author(s).)

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The thick filament is a key component of sarcomeres, the basic units of striated muscle 1 . Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases 2 . Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components., (© 2023. The Author(s).)

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin's motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state 3 ; how titin and cMyBP-C may contribute to length-dependent activation 4 ; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease 5,6 . The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

T-cell-based immunotherapies hold tremendous potential in the fight against cancer, thanks to their capacity to specifically targeting diseased cells. Nevertheless, this potential has been tempered with safety concerns regarding the possible recognition of unknown off-targets displayed by healthy cells. In a notorious example, engineered T-cells specific to MAGEA3 (EVDPIGHLY) also recognized a TITIN-derived peptide (ESDPIVAQY) expressed by cardiac cells, inducing lethal damage in melanoma patients. Such off-target toxicity has been related to T-cell cross-reactivity induced by molecular mimicry. In this context, there is growing interest in developing the means to avoid off-target toxicity, and to provide safer immunotherapy products. To this end, we present CrossDome, a multi-omics suite to predict the off-target toxicity risk of T-cell-based immunotherapies. Our suite provides two alternative protocols, i) a peptide-centered prediction, or ii) a TCR-centered prediction. As proof-of-principle, we evaluate our approach using 16 well-known cross-reactivity cases involving cancer-associated antigens. With CrossDome, the TITIN-derived peptide was predicted at the 99+ percentile rank among 36,000 scored candidates (p-value < 0.001). In addition, off-targets for all the 16 known cases were predicted within the top ranges of relatedness score on a Monte Carlo simulation with over 5 million putative peptide pairs, allowing us to determine a cut-off p-value for off-target toxicity risk. We also implemented a penalty system based on TCR hotspots, named contact map (CM). This TCR-centered approach improved upon the peptide-centered prediction on the MAGEA3-TITIN screening (e.g., from 27th to 6th, out of 36,000 ranked peptides). Next, we used an extended dataset of experimentally-determined cross-reactive peptides to evaluate alternative CrossDome protocols. The level of enrichment of validated cases among top 50 best-scored peptides was 63% for the peptide-centered protocol, and up to 82% for the TCR-centered protocol. Finally, we performed functional characterization of top ranking candidates, by integrating expression data, HLA binding, and immunogenicity predictions. CrossDome was designed as an R package for easy integration with antigen discovery pipelines, and an interactive web interface for users without coding experience. CrossDome is under active development, and it is available at https://github.com/AntunesLab/crossdome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fonseca and Antunes.)

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle) 1-8 . Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle 9-11 . However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation 12 . Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements 13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The discovery of the giant protein titin, also known as connectin, dates almost half a century back. In this review, I recapitulate major advances in the discovery of the titin filaments and the recognition of their properties and function until today. I briefly discuss how our understanding of the layout and interactions of titin in muscle sarcomeres has evolved and review key facts about the titin sequence at the gene (TTN) and protein levels. I also touch upon properties of titin important for the stability of the contractile units and the assembly and maintenance of sarcomeric proteins. The greater part of my discussion centers around the mechanical function of titin in skeletal muscle. I cover milestones of research on titin's role in stretch-dependent passive tension development, recollect the reasons behind the enormous elastic diversity of titin, and provide an update on the molecular mechanisms of titin elasticity, details of which are emerging even now. I reflect on current knowledge of how muscle fibers behave mechanically if titin stiffness is removed and how titin stiffness can be dynamically regulated, such as by posttranslational modifications or calcium binding. Finally, I highlight novel and exciting, but still controversially discussed, insight into the role titin plays in active tension development, such as length-dependent activation and contraction from longer muscle lengths., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the relationship between the muscle shear modulus of the biceps brachii, urinary titin N-terminal fragment (UTF), and other damage markers after eccentric exercise. Seventeen healthy males performed five sets of ten eccentric exercises with dumbbells weighing 50% of the maximum voluntary contraction (MVC) at the elbow joint. Muscle shear modulus with range of interest set to only biceps brachii muscle measured by ultrasound shear wave elastography, UTF, MVC, range of motion (ROM), and soreness (SOR) were recorded before, immediately after, and 1, 24, 48, 72, 96, and 168 h after eccentric exercise. Each marker changed in a time course pattern, as found in previous studies. The peak shear modulus showed a moderate negative correlation with peak MVC (r = -0.531, P < 0.05) and a strong positive correlation with peak UTF (r = 0.707, P < 0.01). Our study results revealed a significant relationship between muscle strength, shear modulus measured by ultrasound SWE, and titin measured by UTF, as a non-invasive damage marker after eccentric exercise to track changes in EIMD., (© Journal of Sports Science and Medicine.)

Titin is the largest protein in humans, composed of more than one hundred immunoglobulin (Ig) domains, and plays a critical role in muscle's passive elasticity. Thus, the molecular design of this giant polyprotein is responsible for its mechanical function. Interestingly, most of these Ig domains are connected directly with very few interdomain residues/linker, which suggests such a design is necessary for its mechanical stability. To understand this design, we chose six representative Ig domains in titin and added nine glycine residues (9G) as an artificial interdomain linker between these Ig domains. We measured their mechanical stabilities using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) and compared them to the natural sequence. The AFM results showed that the linker affected the mechanical stability of Ig domains. The linker mostly reduces its mechanical stability to a moderate extent, but the opposite situation can happen. Thus, this effect is very complex and may depend on each particular domain's property.

Biopolymers, such as polynucleotides, polypeptides and polysaccharides, are macromolecules that direct most of the functions in living beings. Studying the mechanical unfolding of biopolymers provides important information about their molecular elasticity and mechanical stability, as well as their energy landscape, which is especially important in proteins, since their three-dimensional structure is essential for their correct activity. In this primer, we present how to study the mechanical properties of proteins with atomic force microscopy and how to obtain information about their stability and energetic landscape. In particular, we discuss the preparation of polyprotein constructs suitable for AFM single molecule force spectroscopy (SMFS), describe the parameters used in our force-extension SMFS experiments and the models and equations employed in the analysis of the data. As a practical example, we show the effect of the temperature on the unfolding force, the distance to the transition state, the unfolding rate at zero force, the height of the transition state barrier, and the spring constant of the protein for a construct containing nine repeats of the I27 domain from the muscle protein titin. HIGHLIGHTS: 1. Atomic force microscopy (AFM) can be used to study the mechanical unfolding of polymers. 2. AFM provides a direct measurement of unfolding (unbinding) forces. 3. Force measurements for different rates provide information about the distance to the transition state and the unfolding rate at zero force., (© 2022 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Titin, the largest muscle protein, plays an important role in passive tension, sarcomeric integrity and cell signaling within the muscle. Recent work has also highlighted a role for titin in active muscle and the N2A region found in skeletal muscle titin and in some isoforms of cardiac titin has been linked to this function. The N2A region is a multi-domain region composed of four immunoglobulin domains (I80-I83) and a disordered region called the insertion sequence. Previously, our lab has shown that the N2A region binds F-actin in a calcium dependent manner, but it is not known which domains within this region are critical for this binding to occur. In this work, we have used co-sedimentation to demonstrate that only constructs containing the I80 domain are capable of binding F-actin. In addition, binding was only observed in constructs containing at least 3 immunoglobulin domains suggesting a length-dependence to binding. Finally, the calcium-dependence of N2A binding is lost when I83 is not present, consistent with the calcium stabilization that has been reported for this domain. Based on these results, we propose that I80 is critical for initiating binding to F-actin and that I83 is responsible for the calcium dependence., Competing Interests: Declaration of competing interest The authors do not have any conflict of interests associated with this project., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mutations in the titin (TTN) gene are among the most common genomic aberrations in ocular surface squamous neoplasia (OSSN), the most common cancer of the external eye. Further, TTN mutations are associated with resistance to standard therapy with topical interferon alpha-2b (IFN-α2b). However, it remains unclear how TTN mutations drive OSSN pathogenesis and treatment resistance. TTN encodes the largest protein in the human body and its best understood function is as a myofibril scaffold in striated muscle. However, recent evidence indicates that TTN has additional functions in non-muscle cells and in cancer. Here, we performed a disorder-based bioinformatics analysis which revealed that intrinsically disordered protein regions are abundant in TTN and provide mechanistic insights into its function as a nuclear protein in epithelial cells. Specific mutations found in OSSN are predicted to affect its intrinsically disordered protein regions (IDPRs), promoting chromosomal instability, oncogenesis, and altered response to IFN-α2b treatment., (Copyright © 2021. Published by Elsevier B.V.)

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete's heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin's role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete's heart. The athlete's heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete's heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

Manmade high-performance polymers are typically non-biodegradable and derived from petroleum feedstock through energy intensive processes involving toxic solvents and byproducts. While engineered microbes have been used for renewable production of many small molecules, direct microbial synthesis of high-performance polymeric materials remains a major challenge. Here we engineer microbial production of megadalton muscle titin polymers yielding high-performance fibers that not only recapture highly desirable properties of natural titin (i.e., high damping capacity and mechanical recovery) but also exhibit high strength, toughness, and damping energy - outperforming many synthetic and natural polymers. Structural analyses and molecular modeling suggest these properties derive from unique inter-chain crystallization of folded immunoglobulin-like domains that resists inter-chain slippage while permitting intra-chain unfolding. These fibers have potential applications in areas from biomedicine to textiles, and the developed approach, coupled with the structure-function insights, promises to accelerate further innovation in microbial production of high-performance materials., (© 2021. The Author(s).)

Our insight into the diverse and complex nature of dilated cardiomyopathy (DCM) genetic architecture continues to evolve rapidly. The foundations of DCM genetics rest on marked locus and allelic heterogeneity. While DCM exhibits a Mendelian, monogenic architecture in some families, preliminary data from our studies and others suggests that at least 20% to 30% of DCM may have an oligogenic basis, meaning that multiple rare variants from different, unlinked loci, determine the DCM phenotype. It is also likely that low-frequency and common genetic variation contribute to DCM complexity, but neither has been examined within a rare variant context. Other types of genetic variation are also likely relevant for DCM, along with gene-by-environment interaction, now established for alcohol- and chemotherapy-related DCM. Collectively, this suggests that the genetic architecture of DCM is broader in scope and more complex than previously understood. All of this elevates the impact of DCM genetics research, as greater insight into the causes of DCM can lead to interventions to mitigate or even prevent it and thus avoid the morbid and mortal scourge of human heart failure.

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Various amyloid aggregates, in particular, aggregates of amyloid β-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG) n , n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We measured viscoelasticity of two nanoscale systems, single protein molecules and molecular layers of water confined between solid walls. In order to quantify the viscoelastic response of these nanoscale systems in liquid environment, the measurements are performed using two types of atomic force microscopes (AFMs), which employ different detection schemes to measure the cantilever response. We used a deflection detection scheme, available in commercial AFMs, that measures cantilever bending and a fibre-interferometer based detection which measures cantilever displacement. The hydrodynamics of the cantilever is modelled using Euler-Bernoulli equation with appropriate boundary conditions which accommodate both detection schemes. In a direct contradiction with many reports in the literature, the dissipation coefficient of a single octomer of titin I27 8 is found to be immeasurably low. The upper bound on the dissipation coefficient is 5 × 10 -7 kg s -1 , which is much lower than the reported values. The entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN m -1 . We show that in a conventional deflection detection measurement, the phase of the bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which has negligibly small phase lag due to hydrodynamics of the cantilever at low excitation frequencies, is better suited for ensuring artefact-free measurement of viscoelasticity compared to the measurement of the cantilever bending. Further, it was possible to measure dissipation in molecular layers of water confined between the tip and the substrate using fibre interferometer based AFM with similar experimental parameters. It confirms that the dissipation coefficient of a single I27 8 is below the detection limit of AFM. The results shed light on the discrepancy observed in the measured diffusional dynamics of protein collapse measured using Force spectroscopic techniques and single-molecule optical techniques.

Discovery of blood biomarkers to evaluate exercise-induced muscle damage have attracted many researchers and coaches. This study aimed to determine changes in circulating myomesin 3 fragments as a novel biomarker for exercise-induced muscle damage. Nine healthy males performed 10 sets of 40 repetitions of one-leg calf-raise exercise by the load corresponding to the half of their body weight. Muscle symptoms were evaluated by a visual analog scale (VAS). Blood samples were collected before and 2, 4, 24, 48, 72, and 96 h post-exercise. Plasma myomesin 3 fragments levels were significantly increased at 96 h after the eccentric exercise. The myomesin 3 fragments levels were correlated with other biomarkers of muscle damage and the muscle symptoms. These results suggest that the circulating myomesin 3 fragments levels are potential biomarkers reflecting eccentric exercise-induced muscle damage.

FBXL21 is a clock-controlled E3 ligase modulating circadian periodicity via subcellular-specific CRYPTOCHROME degradation. How FBXL21 regulates tissue-specific circadian physiology and what mechanism operates upstream is poorly understood. Here we report the sarcomere component TCAP as a cytoplasmic substrate of FBXL21. FBXL21 interacts with TCAP in a circadian manner antiphasic to TCAP accumulation in skeletal muscle, and circadian TCAP oscillation is disrupted in Psttm mice with an Fbxl21 hypomorph mutation. GSK-3β phosphorylates FBXL21 and TCAP to activate FBXL21-mediated, phosphodegron-dependent TCAP degradation. GSK-3β inhibition or knockdown diminishes FBXL21-Cul1 complex formation and delays FBXL21-mediated TCAP degradation. Finally, Psttm mice show significant skeletal muscle defects, including impaired fiber size, exercise tolerance, grip strength, and response to glucocorticoid-induced atrophy, in conjunction with cardiac dysfunction. These data highlight a circadian regulatory pathway where a GSK-3β-FBXL21 functional axis controls TCAP degradation via SCF complex formation and regulates skeletal muscle function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.

Single-molecule force spectroscopy techniques allow for the measurement of several static and dynamic features of macromolecules of biological origin. In particular, atomic force microscopy, used with a variable pulling rate, provides valuable information on the folding/unfolding dynamics of proteins. We propose here two different models able to describe the out-of-equilibrium statistical mechanics of a chain composed of bistable units. These latter represent the protein domains, which can be either folded or unfolded. Both models are based on the Langevin approach and their implementation allows for investigating the effect of the pulling rate and of the device intrinsic elasticity on the chain unfolding response. The theoretical results (both analytical and numerical) have been compared with experimental data concerning the unfolding of the titin and filamin proteins, eventually obtaining a good agreement over a large range of the pulling rates.

The giant protein titin is expressed in vertebrate striated muscle where it spans half a sarcomere from the Z-disc to the M-band and is essential for muscle organisation, activity and health. The C-terminal portion of titin is closely associated with the thick, myosin-containing filament and exhibits a complex pattern of immunoglobulin and fibronectin domains. This pattern reflects features of the filament organisation suggesting that it acts as a molecular ruler and template, but the exact axial disposition of the molecule has not been determined. Here, we present data that allow us to precisely locate titin domains axially along the thick filament from its tip to the edge of the bare zone. We find that the domains are regularly distributed along the filament at 4-nm intervals and we can determine the domains that associate with features of the filament, such as the 11 stripes of accessory proteins. We confirm that the nine stripes ascribed to myosin binding protein-C are not related to the titin sequence previously assumed; rather, they relate to positions approximately 18 domains further towards the C terminus along titin. This disposition also allows a subgroup of titin domains comprising two or three fibronectin domains to associate with each of the 49 levels of myosin heads in each half filament. The results strongly support the role of titin as a blueprint for the thick filament and the arrangement of the myosin motor domains., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this paper, the property of the muscle titin protein to form in vitro specific amyloid-like aggregates is discussed. The main difference from the known amyloid aggregates is the formation of a quaternary structure that resembles cross-β, with no changes in the secondary structure. Based on the results obtained earlier, as well as the results of this study, we make assumptions about changes in the structure of titin that occur during the formation of amyloid-like aggregates. In particular, our X-ray diffraction data on the titin aggregates suggest that β-strands in the aggregates of this protein are not located perpendicular to the fibril axis, as described for other amyloid proteins, but in parallel. The distance between the β-sheets in the aggregates may vary, and the β-sheets themselves are not strictly oriented along one of the axes, which can lead to the appearance of a diffuse ring reflection of ~8-12 Å. In this regard, the titin aggregates should not be called amyloid, but amyloid-like, with a quaternary structure that resembles cross-β. It cannot be excluded that the formation of this quaternary structure can also occur due to the partial unfolding of titin domains, followed by the interaction of open β-strands between neighboring domains and/or domains of neighboring molecules.

Describing the whole story of protein folding is currently the main enigmatic problem in molecular bioinformatics study. Protein folding mechanisms have been intensively investigated with experimental as well as simulation techniques. Since a protein folds into its specific 3D structure from a unique amino acid sequence, it is interesting to extract as much information as possible from the amino acid sequence of a protein. Analyses based on inter-residue average distance statistics and a coarse-grained Gō-model simulation were conducted on Ig and FN3 domains of a titin protein to decode the folding mechanisms from their sequence data and native structure data, respectively. The central region of all domains was predicted to be an initial folding unit, that is, stable in an early state of folding. This common feature coincides well with the experimental results and underscores the significance of the β-sandwich proteins' common structure, namely, the key strands for folding and the Greek-key motif, which is located in the central region. We confirmed that our sequence-based techniques were able to predict the initial folding event just next to the denatured state and that a 3D-based Gō-model simulation can be used to investigate the whole process of protein folding., (© 2019 Wiley Periodicals, Inc.)

Molecular mechanics force fields have been shown to differ in their predictions of biomolecular processes such as protein folding. To test how force field differences affect predicted polypeptide behavior, we created a mechanically perturbed model of the β-stranded I91 titin domain based on atomic force spectroscopy data and examined its refolding behavior using six different force fields. We found that different force fields varied significantly in their ability to refold the mechanically perturbed I91 domain. Examination of the perturbed I91 unfolded state revealed that all five Amber force fields oversample a specific region of the Ramachandran plot, thereby creating unfolded state intermediates which are not predicted by the Charmm 22* force field. Simulations of perturbed I91 refolding with Amber FB15 revealed that Amber FB15 destabilizes stable portions of I91, thereby contradicting experimental stability analyses. Finally, inspection of the perturbed I91 unfolded state along with equilibration simulations of the Ac-(AAQAA) 3 -NH 2 peptide suggest that high dihedral torsional barriers cause the Amber ff14SB force field to predict higher helical lifetimes relative to other force fields. These results suggest that using mechanically perturbed models can provide a controlled method to gain insights into how force fields affect polypeptide behavior.

Titin is a large filamentous protein that spans half a sarcomere, from Z-disk to M-line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein-protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80-I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81-I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔG unfolding , which is increased in the presence of calcium (pCa 4.3), indicating that Ca 2+ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca 2+ might influence binding events involving N2A., (© 2020 The Protein Society.)

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal β-sheet structure of monovalent streptavidin's functional subunit. Based on these results, we now introduced two mutations (T18C,A33C) in the functional subunit of monovalent streptavidin to establish a switchable connection (disulfide bridge) between the first two β-strands to prevent this unfolding. In atomic force microscopy-based single-molecule force spectroscopy experiments, we observed unbinding forces of about 350 pN (at a force-loading rate of 10 nN s -1 ) for pulling a single biotin out of an N-terminally anchored monovalent streptavidin binding pocket - about 1.5-fold higher compared with what has been reported for N-terminal force loading of native monovalent streptavidin. Upon addition of a reducing agent, the unbinding forces dropped back to 200 pN, as the disulfide bridge was destroyed. Switching from reducing to oxidizing buffer conditions, the inverse effect was observed. Our work illustrates how the mechanics of a receptor-ligand system can be tuned by engineering the receptor protein far off the ligand-binding pocket.

The effects of detergent sodium dodecyl sulfate (SDS) on protein structure and dynamics are fundamental to the most common laboratory technique used to separate proteins and determine their molecular weights: polyacrylamide gel electrophoresis. However, the mechanism by which SDS induces protein unfolding and the microstructure of protein-SDS complexes remain largely unknown. Here, we report a detailed account of SDS-induced unfolding of two proteins-I27 domain of titin and β-amylase-obtained through all-atom molecular dynamics simulations. Both proteins were found to spontaneously unfold in the presence of SDS at boiling water temperature on the time scale of several microseconds. The protein unfolding was found to occur via two distinct mechanisms in which specific interactions of individual SDS molecules disrupt the protein's secondary structure. In the final state of the unfolding process, the proteins are found to wrap around SDS micelles in a fluid necklace-and-beads configuration, where the number and location of bound micelles changes dynamically. The global conformation of the protein was found to correlate with the number of SDS micelles bound to it, whereas the number of SDS molecules directly bound to the protein was found to define the relaxation time scale of the unfolded protein. Our microscopic characterization of SDS-protein interactions sets the stage for future refinement of SDS-enabled protein characterization methods, including protein fingerprinting and sequencing using a solid-state nanopore.

Cardiac aging affects the heart on the functional, structural, and molecular level and shares characteristic hallmarks with the development of chronic heart failure. Apart from age-dependent left ventricular hypertrophy and fibrosis that impairs diastolic function, diminished activity of cardiac protein-quality-control systems increases the risk of cytotoxic accumulation of defective proteins. Here, we studied the impact of cardiac aging on the sarcomeric protein titin by analyzing titin-based cardiomyocyte passive tension, titin modification and proteasomal titin turnover. We analyzed left ventricular samples from young (6 months) and old (20 months) wild-type mice and healthy human donor patients grouped according to age in young (17-50 years) and aged hearts (51-73 years). We found no age-dependent differences in titin isoform composition of mouse or human hearts. In aged hearts from mice and human we determined altered titin phosphorylation at serine residues S4010 and S4099 in the elastic N2B domain, but no significant changes in phosphorylation of S11878 and S12022 in the elastic PEVK region. Importantly, overall titin-based cardiomyocyte passive tension remained unchanged. In aged hearts, the calcium-activated protease calpain-1, which provides accessibility to ubiquitination by releasing titin from the sarcomere, showed decreased proteolytic activity. In addition, we observed a reduction in the proteasomal activities. Taken together, our data indicate that cardiac aging does not affect titin-based passive properties of the cardiomyocytes, but impairs protein-quality control, including titin, which may result in a diminished adaptive capacity of the aged myocardium., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We compare steps observed during the fibrillogenesis of myofibrils with the sequence of steps predictable by a recent analysis of the structurization and functioning of striated muscles. The predicted assembly steps are based solely on fundamental equilibrium processes, particularly supramolecular interactions and liquid crystalline alignment of the rigid thick and thin filaments hosted within the sarcomer. Satisfactory agreement is obtained between several of the observed and the predicted fibrillogenesis steps. In several cases, however, the actual steps appear to be more complex than expected, evidencing the occurrence of transport and kinetic pathways that may assist the attainment of the equilibrium structure. The memory of the order of a precursor mesophase is imprinted during the remodeling of the surfaces at which the two sets of filaments are anchored. The relevance of the present analysis to the functioning of the myofibril is considered.

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2-7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses. Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum.

In striated muscles, molecular filaments are largely composed of long protein chains with extensive arrays of identically folded domains, referred to as "beads-on-a-string". It remains a largely unresolved question how these domains have developed a unique molecular profile such that each carries out a distinct function without false-positive readout. This study focuses on the M-band segment of the sarcomeric protein titin, which comprises ten identically folded immunoglobulin domains. Comparative analysis of high-resolution structures of six of these domains ‒ M1, M3, M4, M5, M7, and M10 ‒ reveals considerable structural diversity within three distinct loops and a non-conserved pattern of exposed cysteines. Our data allow to structurally interpreting distinct pathological readouts that result from titinopathy-associated variants. Our findings support general principles that could be used to identify individual structural/functional profiles of hundreds of identically folded protein domains within the sarcomere and other densely crowded cellular environments., Competing Interests: The authors have declared that no competing interests exist.

Softening is always a problem in fish preservation. This study was aimed to investigate the role of myofibrillar structural proteins degradation in fish softening. The changes of myofibrillar structural proteins, muscle ultrastructure, myofibril fragmentation, and shear force were studied. The results indicated that during the superchilled preservation of grass carp (Ctenopharyngodon idella), small (low-molecular-weight) myofibrillar structural proteins like desmin and troponin-T initiated textural deterioration, leading to Z-disk weakening and actin loosening. In contrast, giant (high-molecular-weight) myofibrillar structural proteins like titin and nebulin were degraded in more amount in the later storage, contributing to Z-disk and M-band disassembly and vague of light and dark regions (I and A bands). Compared to each other, desmin and titin played more important part in softening. All these changes were involved in the increase of muscle fibril segments and the sharp decrease of shear force., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene ( CAPN3 ). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10 13 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Muscle contraction is initiated by the interaction between actin and myosin filaments. The sliding of actin filaments relative to myosin filaments is produced by cross-bridge cycling, which is governed by the theoretical framework of the cross-bridge theory. The cross-bridge theory explains well a number of mechanical responses, such as isometric and concentric contractions. However, some experimental observations cannot be explained with the cross-bridge theory; for example, the increased isometric force after eccentric contractions. The steady-state, isometric force after an eccentric contraction is greater than that attained in a purely isometric contraction at the same muscle length and same activation level. This well-acknowledged and universally observed property is referred to as residual force enhancement (rFE). Since rFE cannot be explained by the cross-bridge theory, alternative mechanisms for explaining this force response have been proposed. In this review, we introduce the basic concepts of sarcomere length non-uniformity and titin elasticity, which are the primary candidates that have been used for explaining rFE, and discuss unresolved problems regarding these mechanisms, and how to proceed with future experiments in this exciting area of research.

The reversible unfolding-refolding transition is considerably important for natural elastomeric proteins (e.g., titin) to fulfill their biological functions. It is of great importance to develop synthetic versions by borrowing their unique stretchable design principles. Herein, we present a novel pulsating vesicle by means of the aqueous self-assembly of supra-amphiphilic helices. Interestingly, this vesicle simultaneously features dynamic swelling and shrinkage movements in response to external proton triggers. Titin-like unfolding-refolding transformation of artificial helices was proved to play a crucial role in this pulsatile motion. Moreover, the vesicular membrane of this vesicle has exhibited tunable permeability during reversible expansion and contraction circulation. Meanwhile, light can also be used as a driving force to further regulate the disassembly-reassembly transformation of the pulsating vesicle. In addition, the drug delivery system was also employed as an investigating model to estimate the permeability variation and disassembly-reassembly behaviors of the pulsating vesicles, which displayed unique dual quick- and sustained-release behaviors toward anti-cancer agents. It is anticipated that this work opens an avenue for fabricating novel stretchable biomimetics by using the exclusive unfolding-refolding nature of artificial foldamers.

The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Cerebral palsy (CP) is the result of a static brain lesion which causes spasticity and muscle contracture. The source of the increased passive stiffness in patients is not understood and while whole muscle down to single muscle fibres have been investigated, the smallest functional unit of muscle (the sarcomere) has not been. Muscle biopsies (adductor longus and gracilis) from pediatric patients were obtained (CP n = 9 and control n = 2) and analyzed for mechanical stiffness, in-vivo sarcomere length and titin isoforms. Adductor longus muscle was the focus of this study and the results for sarcomere length showed a significant increase in length for CP (3.6 µm) compared to controls (2.6 µm). Passive stress at the same sarcomere length for CP compared to control was significantly lower in CP and the elastic modulus for the physiological range of muscle was lower in CP compared to control (98.2 kPa and 166.1 kPa, respectively). Our results show that CP muscle at its most reduced level (the myofibril) is more compliant compared to normal, which is completely opposite to what is observed at higher structural levels (single fibres, muscle fibre bundles and whole muscle). It is noteworthy that at the in vivo sarcomere length in CP, the passive forces are greater than normal, purely as a functional of these more compliant sarcomeres operating at long lengths. Titin isoforms were not different between CP and non-CP adductor longus but titin:nebulin was reduced in CP muscle, which may be due to titin loss or an over-expression of nebulin in CP muscles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The protein titin plays a key role in vertebrate muscle where it acts like a giant molecular spring. Despite its importance and conservation over vertebrate evolution, a lack of high quality annotations in non-model species makes comparative evolutionary studies of titin challenging. The PEVK region of titin-named for its high proportion of Pro-Glu-Val-Lys amino acids-is particularly difficult to annotate due to its abundance of alternatively spliced isoforms and short, highly repetitive exons. To understand PEVK evolution across mammals, we developed a bioinformatics tool, PEVK&#95;Finder, to annotate PEVK exons from genomic sequences of titin and applied it to a diverse set of mammals. PEVK&#95;Finder consistently outperforms standard annotation tools across a broad range of conditions and improves annotations of the PEVK region in non-model mammalian species. We find that the PEVK region can be divided into two subregions (PEVK-N, PEVK-C) with distinct patterns of evolutionary constraint and divergence. The bipartite nature of the PEVK region has implications for titin diversification. In the PEVK-N region, certain exons are conserved and may be essential, but natural selection also acts on particular codons. In the PEVK-C, exons are more homogenous and length variation of the PEVK region may provide the raw material for evolutionary adaptation in titin function. The PEVK-C region can be further divided into a highly repetitive region (PEVK-CA) and one that is more variable (PEVK-CB). Taken together, we find that the very complexity that makes titin a challenge for annotation tools may also promote evolutionary adaptation., (Copyright © 2019 Muenzen et al.)

To understand the folding mechanism of a protein is one of the goals in bioinformatics study. Nowadays, it is enigmatic and difficult to extract folding information from amino acid sequence using standard bioinformatics techniques or even experimental protocols which can be time consuming. To overcome these problems, we aim to extract the initial folding unit for titin protein (Ig and fnIII domains) by means of inter-residue average distance statistics, Average Distance Map (ADM) and contact frequency analysis (F-value). TI I27 and TNfn3 domains are used to represent the Ig-domain and fnIII-domain, respectively. Beta-strands 2, 3, 5, and 6 are significant for the initial folding processes of TI I27. The central strands of TNfn3 were predicted as a primary folding segment. Known 3D structure and unknown 3D structure domains were investigated by structure or non-structure based multiple sequence alignment, respectively, to learn the conserved hydrophobic residues and predicted compact region relevant to evolution. Our results show good correspondence to experimental data, phi-value and protection factor from H-D exchange experiments. The significance of conserved hydrophobic residues near F-value peaks for structural stability using hydrophobic packing is confirmed. Our prediction methods once again could extract a folding mechanism only knowing the amino acid sequence., (© 2018 Wiley Periodicals, Inc.)

The nanomechanics of sarcomeric proteins is a key contributor to the mechanical output of muscle. Among them, titin emerges as a main target for the regulation of the stiffness of striated muscle. In the last years, single-molecule experiments by Atomic Force Microscopy (AFM) have demonstrated that redox posttranslational modifications are strong modulators of the mechanical function of titin. Here, we provide an overview of the recent development of the redox mechanobiology of titin, and suggest avenues of research to better understand how the stiffness of molecules, cells and tissues are modulated by redox signaling in health and disease., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

A synthetic data set demonstrating a particularly challenging case of indexing ambiguity in the context of radiation damage was generated. This set shall serve as a standard benchmark and reference point for the ongoing development of new methods and new approaches to robust structure solution when single-crystal methods are insufficient. Of the 100 short wedges of data, only the first 36 are currently necessary to solve the structure by `cheating', or using the correct reference structure as a guide. The total wall-clock time and number of crystals required to solve the structure without cheating is proposed as a metric for the efficacy and efficiency of a given multi-crystal automation pipeline., (open access.)