Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements

Watanabe, Kaori; Nair, Preetha; Labeit, D.; Kellermayer, Miklós; Greaser, Marion L.; Labeit, Siegfried; Granzier, Henk

doi:10.1074/jbc.m200356200

Cited by 134 publications

(152 citation statements)

References 41 publications

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“…The midpoint force in PullT7 (Fig. 3D) now shifts from 3.5 to 13 pN, showing that the α-actinin/T7 bond can, indeed, resist forces considered physiologically relevant (27)(28)(29). The higher forces for this pulling direction are a consequence of the shorter difference in extension between the bound and the unbound conformations (4 nm of contour length, vs. 34 nm for the PullA-T7 construct, including unfolding of EF3-4): At equilibrium, interaction free energies can be related to the force multiplied by the elongation and the much shorter elongation in the PullT7 construct leads to a higher midpoint unbinding force.…”

Section: Discussionmentioning

confidence: 99%

“…1G). Nonetheless, the midpoint force we measure for the PullA-T7 construct (3.5 pN) is within the physiological range of forces acting on a single titin molecule, estimated to be 0-5 pN in a number of studies (27)(28)(29). The low measured forces in the PullA-T7 construct can be ascribed to the fact that EF3-4 readily unfolds after unbinding of T7, whereas this will not happen if titin and α-actinin are not tethered, as is the case in muscle.…”

Section: Discussionmentioning

confidence: 99%

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α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

Grison

Merkel

Košťan

et al. 2017

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

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Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin crosslinker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.M uscle is the tissue that is constantly subjected to high mechanical loads. Whereas thick and thin filaments are responsible for active force production, the passive elasticity of muscle is dominated by titin/connectin filaments (1). Hence, under passive stretching conditions the integrity of muscle relies on titin's being firmly anchored within the sarcomere, preventing the interdigitated muscle filaments from falling apart (Fig. 1A). Whereas titin is firmly attached to thick filaments in the A-band and the M-line (2-6), it is much less clear how stable anchoring is achieved in the Z-disk, where adjacent sarcomeres overlap. The superstable titin/telethonin interaction within the Z-disk was considered important for titin anchoring (7-9), but knockout mutants later showed that it is not essential for muscle integrity (10-12). Apart from a direct interaction between actin filaments and titin at the Z-disk edge (13), the most prominent candidate for the anchoring of titin within the Z-disk is its interaction with α-actinin (Fig. 1B) (6,12,14).Four isoforms of human α-actinin have been identified: the calcium-insensitive muscle isoforms 2 and 3, which cross-link actin filaments in sarcomere-delimiting Z-disk complexes, and calcium-sensitive nonmuscle isoforms 1 and 4. α-Actinin is an antiparallel homodimer whose most prominent task is crosslinking actin filaments of neighboring sarcomeres in the Z-disk ( Fig. 1B; reviewed in ref. 14). In each subunit, a flexible region called the neck separates the actin binding domain (ABD) from four spectrin-like repeats (SR) forming the rod region (Fig. 1B and Fig. S1). The rod regions of the two subunits interact and provide a rigid spacer between the actin filaments. At the other end of each subunit a calmodulin-like domain (CaMD) formed by two pairs of EF-hands (EF1-2 and EF3-4) is able to bind a Z-disk region of titin formed by the so-called Z-repeats (15-17). The current model for α-actinin 2 dynamic regulation suggests that EF3-4 hands of one subunit bind to the neck region of the juxtaposed subunit, thus not being available for the interaction with titin Z-repeats (Fig. S1) (18, 19). Upon activ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

Grison

Merkel

Košťan

et al. 2017

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

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“…The unique cardiacspecific N2B and PEVK regions are 572 and 186 amino acids long, respectively. The mechanical properties of these regions were recently examined by single molecule AFM techniques and were found to behave like extensible random coils (7)(8)(9). In contrast to mechanically stable folded proteins like the immunoglobulin domains of titin (20), an unstructured protein domain generates a recoiling force that changes monotonically with its length (8).…”

Section: Discussionmentioning

confidence: 99%

The Elasticity of Individual Titin PEVK Exons Measured by Single Molecule Atomic Force Microscopy

Sarkar

Caamano

Fernández

2005

Journal of Biological Chemistry

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The I-band region of the giant muscle protein titin contains a large domain enriched for the amino acids proline, glutamate, valine, and lysine and is denoted the PEVK domain. The PEVK domain of titin encodes a random coil shown to be an important factor in the passive elasticity of titin. Muscle-specific splicing of 116 PEVK exons encodes this domain. It has been proposed that proline contents determine the elasticity of the PEVK polypeptide, where the individual exons code for "flexibility cassettes." To test this hypothesis, we have measured the elasticity of three distinct polypeptides encoded by individual PEVK exons (161, 120 and 184) that varied greatly in their proline contents (7, 14, and 37% respectively) and total PEVK contents (55, 70, and 87%). We used single molecule atomic force microscopy techniques to measure the persistence length, p, of the engineered PEVK proteins. Surprisingly, all three exons 161, 120, and 184 coded for proteins with similar values of persistence length, p ‫؍‬ 0.92 ؎ 0.38, 0.89 ؎ 0.42, and 0.98 ؎ 0.4 nm, respectively. We conclude that the PEVK exons encode polypeptides of similar elastic properties, unrelated to their total PEVK contents. Hence, alternative splicing solely adjusts the length of the PEVK domain of titin.

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“…Those studies of intact titin and the Ig/Fn3 domains have provided a deeper understanding of how the elasticity of titin is adjusted by the sequential unfolding of Ig/Fn3 domains and how its elasticity recovers after domain refolding (Kellermayer et al, 1997;Rief et al, 1997;Carrion-Vazquez et al, 2000). More recently, the mechanical properties of expressed PEVK fragments based on cardiac and soleus PEVK exons were reported Linke et al, 2002;Watanabe et al, 2002;Labeit et al, 2003;Sarkar et al, 2005). Each study observed an unexpectedly broad distribution in the measured elastic persistence length of the PEVK segment.…”

Section: Introductionmentioning

confidence: 99%

Titin PEVK segment: charge-driven elasticity of the open and flexible polyampholyte

Forbes¹,

Jin²,

Ma³

et al. 2006

J Muscle Res Cell Motil

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The giant protein titin spans half of the sarcomere length and anchors the myosin thick filament to the Z-line of skeletal and cardiac muscles. The passive elasticity of muscle at a physiological range of stretch arises primarily from the extension of the PEVK segment, which is a polyampholyte with dense and alternating-charged clusters. Force spectroscopy studies of a 51 kDa fragment of the human fetal titin PEVK domain (TP1) revealed that when charge interactions were reduced by raising the ionic strength from 35 to 560 mM, its mean persistence length increased from 0.30±0.04 nm to 0.60±0.07 nm. In contrast, when the secondary structure of TP1 was altered drastically by the presence of 40 and 80% (v/v) of trifluoroethanol, its force-extension behavior showed no significant shift in the mean persistence length of ~0.18±0.03 nm at the ionic strength of 15 mM. Additionally, the mean persistence length also increased from 0.29 to 0.41 nm with increasing calcium concentration from pCa 5-8 to pCa 3-4. We propose that PEVK is not a simple entropic spring as is commonly assumed, but a highly evolved, gel-like enthalpic spring with its elasticity dominated by the sequence-specific charge interactions. A single polyampholyte chain may be fine-tuned to generate a broad range of molecular elasticity by varying charge pairing schemes and chain configurations.

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Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements

Cited by 134 publications

References 41 publications

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk

The Elasticity of Individual Titin PEVK Exons Measured by Single Molecule Atomic Force Microscopy

Titin PEVK segment: charge-driven elasticity of the open and flexible polyampholyte

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