Structural changes in the myosin filament and cross‐bridges during active force development in single intact frog muscle fibres: stiffness and X‐ray diffraction measurements

Brunello, Elisabetta; Bianco, Pasquale; Piazzesi, Gabriella; Linari, Marco; Reconditi, Massimo; Panine, Pierre; Narayanan, Theyencheri; Helsby, W.; Irving, Malcolm; Lombardi, Vincenzo

doi:10.1113/jphysiol.2006.115394

Cited by 58 publications

(90 citation statements)

References 40 publications

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“…300 s −1 , slower than the force decrease but equal to the expected rate of myosin head detachment (15). This experiment shows that this component of the change in orientation of the E helix coincides with myosin head binding to actin rather than to active force per se, although during the initial activation following Ca 2+ release force generation follows myosin head binding with a lag of less than 1 ms (30).…”

Section: Discussionmentioning

confidence: 81%

Structural dynamics of troponin during activation of skeletal muscle

Fusi

Brunello

Sevrieva

et al. 2014

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

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Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5-7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca 2+ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca 2+ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg 2+ as well as Ca 2+ . The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the "IT arm" (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lob...

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Section: Discussionmentioning

confidence: 81%

Structural dynamics of troponin during activation of skeletal muscle

Fusi

Brunello

Sevrieva

et al. 2014

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

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“…However, thick filament compliance can have a profound effect on myosin kinetics and the contractile properties of muscle. [3][4][5] Changes in thick filament length during contraction have been documented in vertebrate [6][7][8] and invertebrate 9 muscles, yet the molecular basis of this compliance is not well understood. We showed in a previous study that myosin binding protein C (MyBP-C), a protein that binds to the myosin rod, contributes to the stiffness of mouse cardiac thick filaments.…”

Section: Introductionmentioning

confidence: 99%

Flightin Is Necessary for Length Determination, Structural Integrity, and Large Bending Stiffness of Insect Flight Muscle Thick Filaments

Contompasis

Nyland

Maughan

et al. 2010

Journal of Molecular Biology

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“…Changes in thick filament length during muscle contraction have been documented in isolated muscles [2][3][4] and intact animals. 5 X-ray diffraction patterns obtained from flying Drosophila revealed time-resolved changes in the spacing of the 7.2-nm reflection that originates from the myosin filament backbone.…”

Section: Introductionmentioning

confidence: 99%