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

Abstract

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 ...

Structural change of crossbridges of rabbit skeletal muscle during isometric contraction

“…However, other in vitro studies reported an angle close to that in rigor (Zot et al, 1990). The EPR studies that showed rotational motion of heads during contraction (Cooke et al, 1982;Barnett & Thomas, 1989;Fajer et al, 1990) are also consistent with our results.…”

“…X-ray diffraction (Huxley & Brown, 1967;Yagi et al, 1981;Huxley et al, 1982) and EPR studies (Cooke et al, 1982;Barnett & Thomas, 1989;Fajer et al, 1990) have suggested that the averaged structure of contracting muscle is different from that of rigor or relaxed muscle. The results of X-ray diffraction were interpreted to mean that during contraction the helical arrangement of myosin heads along thick filaments is disordered but the --, 14.3 nm subunit repeat is maintained.…”

The Structural Dynamics of Actin during Active Interaction with Myosin Depends on the Isoform of the Essential Light Chain

“…(4, 55–59) In the S state, myosin heads form stereospecific contacts with actin, while in the W state the catalytic and light-chain domains of the myosin head show rotational disorder on the microsecond time scale. (4, 24, 55, 58, 60–64) TPA studies with probes on actin have shown that actin also has two predominant structural states, S and W (more dynamically disordered), related to the structural states of the interacting myosin head. (2, 6, 7)…”

Orientational distribution of spin-labeled actin oriented by flow

“…Alternatively, it is possible that the S 1-induced changes in the above studies do not reflect conformational changes in actin but reflect local rotations ofthe probes or their immediate environments (Fajer et al, 1990b). To investigate these possibilities more thoroughly, (a) spin labels should be attached to other sites on actin (e.g., the myosin binding site) or (b) caged ATP studies (Ostap and Thomas, 1991;Fajer et al, 1990c) should be done on oriented actin samples.…”