Flexibility and extensibility in the titin molecule: analysis of electron microscope data

Tskhovrebova, Larissa; Trinick, John

doi:10.1006/jmbi.2001.4700

Cited by 55 publications

(47 citation statements)

References 90 publications

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“…This points to the existence of an architectural and dynamic supra-pattern in the filament caused by a regularity of molecular features. Speculatively, the inferred filament pattern might correspond to the observation of local pseudohelical substructure in EM (17) and atomic force microscopy (AFM) (18) studies of full-length titin molecules.…”

Section: Conservation Of Intermodular Transitionmentioning

confidence: 70%

A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

Castelmur

Marino

Svergun

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. To date, the molecular events underlying the mechanics of the folded titin chain remain largely unknown. We have elucidated the crystal structure of the 6-Ig fragment I65-I70 from the elastic I-band fraction of titin and validated its conformation in solution using small angle x-ray scattering. The long-range properties of the chain have been visualized by electron microscopy on a 19-Ig fragment and modeled for the full skeletal tandem. Results show that conserved Ig-Ig transition motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension. There, librational entropy can be expected to act as an energy barrier to prevent Ig unfolding while, instead, triggering the unraveling of flanking springs formed by proline, glutamate, valine, and lysine (PEVK) sequences. We propose a mechanistic model based on freely jointed rigid segments that rationalizes the response to stretch of titin Ig-tandems according to molecular features.electron microscopy ͉ poly-Ig tandem structure ͉ small angle x-ray scattering ͉ titin elasticity ͉ x-ray crystallography T he striated muscle of vertebrate is characterized by a striking elasticity that allows it to store mechanical energy and stretch over twice its resting length without disrupting its structural integrity. At physiological amounts of stretch, most of the elastic response of the myofibril is generated by the intrasarcomeric titin filament (Ϸ3.2 MDa, Ͼ1-m length). This protein functions as a bidirectional spring that stretches and recoils during muscle function to return the myofibril to its resting length (1). The spring components of titin are located in its I-band fraction, which forms an elastic connection between the ends of the thick filaments and the Z-disk. Titin contains two main elastic components, a prolinerich PEVK (proline, glutamate, valine, and lysine) segment of up to 2,200 residues length and a poly-Ig array formed by up to 95 modules (2). Both segments straighten upon myofibril stretch developing a passive entropic tension in the sarcomere. Poly-Ig arrays extend at low force whereas PEVK-repeats unravel at higher load, with the combined action of both springs defining the mechanical stiffness of the sarcomere (1, 3). The importance of stretch-recoil control in muscle function is emphasized by the finely tuned composition of both poly-Ig and PEVK segments in titin, which through splicing undergo an extensive adaptation to the different physiological and pathological states of muscle.The molecular basis of titin chain elasticity is currently unknown. The response to stretch of i...

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Section: Conservation Of Intermodular Transitionmentioning

confidence: 70%

A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

Castelmur

Marino

Svergun

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, the model predicts that the unfolding of globular domains occurs even before complete PEVK extension. To test for this possibility, we stretched titin molecules with receding meniscus Tskhovrebova and Trinick, 2001) and visualized their highresolution structure with AFM ( Fig. 7B) (Mártonfalvi and Kellermayer, 2014).…”

Section: Titin As Mechanosensormentioning

confidence: 99%

“…Titin molecules stretched with receding meniscus Tskhovrebova and Trinick, 2001) were imaged with a high-resolution atomic force microscope (Cypher, AsylumResearch, Santa Barbara, CA). Titin was diluted with PBS solution containing 50% glycerol to an approximate final protein concentration of 20 mg/ml.…”

Section: Atomic Force Microscopymentioning

confidence: 99%

Low-force transitions in single titin molecules reflect a memory of contractile history

Mártonfalvi

Bianco

Linari

et al. 2013

Journal of Cell Science

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Titin is a giant elastomeric muscle protein that has been suggested to function as a sensor of sarcomeric stress and strain, but the mechanisms by which it does so are unresolved. To gain insight into its mechanosensory function we manipulated single titin molecules with high-resolution optical tweezers. Discrete, stepwise transitions, with rates faster than canonical Ig domain unfolding occurred during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions (PEVK, proximal tandem-Ig, N2A) are likely to be involved. The pattern of transitions is sensitive to the history of contractile events. Monte-Carlo simulations of our experimental results predicted that structural transitions begin before the complete extension of the PEVK domain. Highresolution atomic force microscopy (AFM) supported this prediction. Addition of glutamate-rich PEVK domain fragments competitively inhibited the viscoelastic response in both single titin molecules and muscle fibers, indicating that PEVK domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends by a complex pattern of history-dependent discrete conformational transitions, which, by dynamically exposing ligand-binding sites, could set the stage for the biochemical sensing of the mechanical status of the sarcomere.

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“…A single molecule stretches from the Zdisk (N-terminus) to the M-band (C-terminus) (Fürst et al, 1988;Wang et al, 1991) and titin molecules overlap in the Zand in the M-band (Gregorio et al, 1998, Obermann et al, 1996. The I-band region of titin is partially elastic and involved in passive tension development in muscle (reviewed by Tskhovrebova and Trinick, 2001;Tskhovrebova and Trinick, 2003). The A-band region of titin is thought to act as a molecular ruler and regulates the length of the thick filament (Labeit et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation

Musa

Meek

Gautel

et al. 2006

Journal of Cell Science

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Titin, a multifunctional protein that stretches from the Z-disk to the M-band in heart and skeletal muscle, contains a kinase domain, phosphorylation sites and multiple binding sites for structural and signalling proteins in the M-band. To determine whether this region is crucial for normal sarcomere development, we created mouse embryonic stem cell (ES) lines in which either one or both alleles contained a targeted deletion of the entire M-band-coding region, leaving Z-disk-binding and myosin-filament-binding sites intact. ES cells were differentiated into cardiomyocytes, and myofibrillogenesis investigated by immunofluorescence microscopy. Surprisingly, deletion of one allele did not markedly affect differentiation into cardiomyocytes, suggesting that a single intact copy of the titin gene is sufficient for normal myofibrillogenesis. By contrast, deletion of both alleles resulted in a failure of differentiation beyond an early stage of myofibrillogenesis. Sarcomeric myosin remained in non-striated structures, Z-disk proteins, such as α-actinin, were mainly found in primitive dot-like structures on actin stress fibres, M-band-associated proteins (myomesin, obscurin, Nbr1, p62 and MURF2) remained punctate. These results show that integration of the M-band region of titin is required for myosin filament assembly, M-band formation and maturation of the Z-disk.

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Flexibility and extensibility in the titin molecule: analysis of electron microscope data

Cited by 55 publications

References 90 publications

A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

Low-force transitions in single titin molecules reflect a memory of contractile history

Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation

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