Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Lovell, Stephen J.; Harrington, William F.

doi:10.1016/0022-2836(81)90352-1

Cited by 53 publications

(34 citation statements)

References 44 publications

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“…5a). In this condition-i.e., in the absence of nucleotideessentially all cross-bridges are attached to the actin filament with very high affinity (31,32), meaning that a stiffness increase in rigor can not be attributed to an increased number of strongly attached myosin heads. Instead, the increase in stiffness of the fibers carrying the mutation in the converter domain must rather be caused by a higher stiffness-i.e., a larger resistance to elastic distortion-of the cross-bridges with the Arg719Trp mutation.…”

Section: Fiber Cross-sectional Area and Packing Of Myofibrils Studied Bymentioning

confidence: 99%

Mutation of the myosin converter domain alters cross-bridge elasticity

Köhler

Winkler²,

Schulte³

et al. 2002

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

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Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the ␤-myosin heavy chain. We found that the Arg-719 3 Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48 -59%. Under rigor and relaxing conditions, fiber stiffness was 45-47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself. It is widely accepted that active force and movement generated by muscle fibers result from structural changes in the head domain of the myosin molecule (the cross-bridge) while it is attached to the actin filament. These changes are thought to involve a tilting of the light-chain-binding domain of the myosin head relative to its catalytic domain (1-7). As a result of such structural changes distortion of an elastic element within the actin-attached myosin head allows strain to develop before movement (8). Relief of this strain drives sliding of actin filaments past the myosin filaments, or alternatively, if filament sliding is prevented, active force is generated because of continued strain of the elastic element. Despite the central significance of this concept, however, it remained unclear which part of the actomyosin complex represents the elastic element-i.e., the element that experiences the main elastic distortion while other parts act more like rigid bodies. Neither the known crystal structures of the myosin head nor cryo-electron microscopy with reconstruction of the actomyosin complex have resolved this question. Some authors considered elastic bending of the long, light-chain-binding ␣-helix an obvious candidate (4, 9, 10). The actin-myosin interface (11) or the junction between the lightchain domain and the catalytic domain of the...

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Section: Fiber Cross-sectional Area and Packing Of Myofibrils Studied Bymentioning

confidence: 99%

Mutation of the myosin converter domain alters cross-bridge elasticity

Köhler

Winkler²,

Schulte³

et al. 2002

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

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“…If the small fraction of heads with rigor-like orientation is responsible for this high stiffness, they must have much higher stiffness than in rigor (10). However, ifwe assume that the fraction of rigor stiffness (75-85%) is the fraction of attached heads, then at most 17% (13%/75%) of the attached heads exhibit rigor-like orientation, since all the heads are bound in rigor (22,23). This upper bound could increase to 34% if all attached cross-bridges are single-headed in active muscle, since single-headed cross-bridges have been shown to be as stiff as double-headed cross-bridges under some conditions (9,10).…”

Section: Methodsmentioning

confidence: 99%

Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP.

Fajer

Thomas

1990

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

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To study the orientation of spin-labeled myosin heads in the first few seconds after the production of saturating ATP, we have used a laser flash to photolyze caged ATP during EPR data acquisition. Rabbit psoas muscle fibers were labeled with maleinide spin label, modifying 60% of the myosin heads without impairing muscle fiber biochemical and physiological activity (ATPase and force). The muscle bundles were incubated for 30 min with 5 mM caged ATP prior to the UV flash. The flash, from an excimer laser, liberated 2-3 mM ATP, generating maximum force in the presence of Ca2l and relaxing fully in the absence of Ca2". Control experiments, using fibers decorated with labeled myosin subfragment, showed that the flash liberates sufficient ATP to saturate myosin active sites in all regions of the muscle bundles. To increase the time resolution, and to minimize the time of the contraction, we followed in time the intensity at a single spectral position (P2)~which is associated with the high degree of orientational order in rigor. ATP liberation produced a rapid decrease of P2 with liberation of ATP, indicating a large decrease in orientational order in both relaxation and contraction. This transient was absent when caged AMP was used, ruling out nonspecific effects of the UV flash and subsequent photochemistry. The steady-state level of P2 during contraction was almost as low as that reached in relaxation, although the duration of the steady state was much more brief in contraction. Upon depletion of ATP in contraction, the P2 intensity reverted to the original rigor level, accompanied by development of rigor tension. The steady-state results obtained in the brief contractions induced by caged ATP are quantitatively consistent with those obtained in longer contractions by continuously perfusing fibers with ATP. In isometric contraction, most (88% ± 4%) of the heads are in a population characterized by a high degree of axial disorder, comparable to that observed for all heads in relaxation. Since the stiffness of these fibers in contraction is 80% of the stiffness in rigor, it is likely that most of the heads in this highly disoriented population are attached to actin in contraction and that most actin-attached heads in contraction are in this disoriented population.Electron microscopic observation of different orientations of muscle cross-bridges with respect to the muscle fiber axis in rigor and relaxation spawned the widely discussed "rotating head" hypothesis of muscle contraction (1). It has been postulated that the myosin head forms a cross-bridge by binding to the actin filament at a 900 angle; it then undergoes a conformational change that results in a pivoting motion of the head to a 45°orientation (2). Strain in the cross-bridge is then relieved by a sliding motion of actin and myosin filaments (2, 3). A rigorous confirmation of this hypothesis requires direct evidence for a large (-45°) tilting motion ofthe entire actin-attached myosin head with respect to the muscle fiber axis. Despite a wide range ...

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“…It was therefore important to investigate the question of possible crossbridge heterogeneity by an alternative experimental technique. In this paper we present an examination of this problem by the tryptic digestion method, introduced to study the association of crossbridges with actin in myofibrils by Lovell & Harrington (1981) and first applied to study the effect of ATP and its analogues on actin-myosin interaction by Reisler and his collaborators (Chen & Reisler, 1983. It relies on the fact that in rigor myosin heads are attacked during mild tryptic treatment at a single prote~ise-sensitive site removed at 26 kDa from the N-terminal end of the myosin heavy chain.…”

Section: Introductionmentioning

confidence: 99%

Actin-attached and detached crossbridges in myofibrils: Segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain

Assulin

Borejdo

Flynn

1986

J Muscle Res Cell Motil

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Tryptic digestion of myofibrils was used to assess the interaction of crossbridges with thin filaments in the presence of ATP analogues. The relative amounts of 200 kDa fragment produced by trypsin from myosin heavy chain when the crossbridge is attached to actin, and of 160 kDa fragment produced when the crossbridge is detached from actin, served as a measure of crossbridge-actin interaction. In rigor only the 200 kDa fragment was produced suggesting that a great majority of the crossbridges were strongly attached to actin; in the presence of MgPPi at 0 degrees C only the 160 kDa fragment was finally produced suggesting that eventually all crossbridges detached from actin. In the presence of MgPPi or MgAMPPNP at 25 degrees C both 200 and 160 kDa fragments were present for several minutes after myosin heavy chain had been completely digested, suggesting that two populations of crossbridges (attached and detached) co-existed at the same time within the myofibril. It is concluded that the addition of ATP analogues to muscle does not simply affect the chemical equilibrium of binding of myosin heads to actin but that it causes rapid dissociation of one crossbridge population without significant effect on binding to actin of the remaining crossbridge population.

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Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Cited by 53 publications

References 44 publications

Mutation of the myosin converter domain alters cross-bridge elasticity

Mutation of the myosin converter domain alters cross-bridge elasticity

Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP.

Actin-attached and detached crossbridges in myofibrils: Segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain

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