Cross-bridge behavior in rigor muscle

Properties of the rigor state in muscle can be explained by a simple cross-bridge model, of the type which has been suggested for active muscle, in which detachment of cross-bridges by ATP is excluded. Two attached cross-bridge states, with distinct force vs. distortion relationships, are required, in addition to a detached state, but the attached cross-bridge states in rigor muscle appear to differ significantly from the attached cross-bridge states in active muscle. The stability of the rigor force maintained in muscle under isometric conditions does not require exceptional stability of the attached cross-bridges, if the positions in which attachment of cross-bridges is allowed are limited so that the attachment of cross-bridges in positions which have minimum free energy is excluded. This explanation of the stability of the rigor state may also be applicable to the maintenance of stable rigor waves on flagella.     

Radial equilibrium lengths of actomyosin cross-bridges in muscle.

Radial equilibrium lengths of the weakly attached, force-generating, and rigor cross-bridges are determined by recording their resistance to osmotic compression. Radial equilibrium length is the surface-to-surface distance between myosin and actin filaments at which attached cross-bridges are, on average, radially undistorted. We previously proposed that differences in the radial equilibrium length represent differences in the structure of the actomyosin cross-bridge. Until now the radial equilibrium length had only been determined for various strongly attached cross-bridge states and was found to be distinct for each state examined. In the present work, we demonstrate that weakly attached cross-bridges, in spite of their low affinity for actin, also exert elastic forces opposing osmotic compression, and they are characterized by a distinct radial equilibrium length (12.0 nm vs. 10.5 nm for force-generating and 13.0 nm for rigor cross-bridge). This suggests significant differences in the molecular structure of the attached cross-bridges under these conditions, e.g., differences in the shape of the myosin head or in the docking of the myosin to actin. Thus, the present finding supports our earlier conclusion that there is a structural change in the attached cross-bridge associated with the transition from a weakly bound configuration to the force-generating configuration. The implications for imposing spatial constraints on modeling actomyosin interaction in the filament lattice are discussed.     

Rigor-force producing cross-bridges in skeletal muscle fibers activated by a substoichiometric amount of ATP

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.     

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.     

Cross-bridge action: present views, prospects, and unknowns

When the sliding filament hypothesis was proposed in 1953-1954, existing evidence showed that (1) contributions to tension were given by active sites uniformly distributed within each zone of filament overlap and (2) each site functioned cyclically. These sites were identified by electron microscopy as cross-bridges between the two filaments, formed of the heads of myosin molecules projecting from a thick filament and attaching to a thin filament. The angle of these cross-bridges was found to be different at rest and in rigor, suggesting that the event causing relative motion of the filaments was a change of the angle of the cross-bridges. At first, it seemed likely that the whole cross-bridge rotated about its attachment to actin, but when the atomic structures of actin and myosin were obtained by X-ray crystallography, a possible hinge was found between the "catalytic domain" which attaches to the actin filament and the "light-chain domain" which appears to act as a lever arm. Two attitudes of the lever arm are now well established, the transition between them being driven by a conformational change coupled to some step in the hydrolysis of ATP, but several recent observations suggest that this is not the whole story: a third attitude has been shown by X-ray crystallography; a non-muscle myosin has been shown to produce its working stroke in two steps; and there are suggestions that an additional displacement of the filaments is produced by a change in the attitude of the catalytic domain on the thin filament.     

Structural characterization of the binding of Myosin*ADP*Pi to actin in permeabilized rabbit psoas muscle

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.     

Mathematical simulation of muscle cross-bridge cycle and force-velocity relationship

Muscle contraction underlies many essential functions such as breathing, heart beating, locomotion, regulation of blood pressure, and airway resistance. Active shortening of muscle is the result of cycling of myosin cross-bridges that leads to sliding of myosin filaments relative to actin filaments. In this study, we have developed a computer program that allows us to alter the rates of transitions between any cross-bridge-states in a stochastic cycle. The cross-bridge states within the cycle are divided into six attached (between myosin cross-bridges and actin filaments) states and one detached state. The population of cross-bridges in each of the states is determined by the transition rates throughout the cycle; differential equations describing the transitions are set up as a cyclic matrix. A method for rapidly obtaining steady-state exact solutions for the cyclic matrix has been developed to reduce computation time and avoid the divergence problem associated with numerical solutions. In the seven-state model, two power strokes are assumed for each cross-bridge cycle, one before the release of inorganic phosphate, and one after. The characteristic hyperbolic force-velocity relationship observed in muscle contraction can be reproduced by the model. Deviation from the single hyperbolic behavior at low velocities can be mimicked by allowing the rate of cross-bridge-attachment to vary with velocity. The effects of [ATP], [ADP], and [P(i)] are simulated by changing transition rates between specific states. The model has revealed new insights on how the force-velocity characteristics are related to the state transitions in the cross-bridge cycle.     

Mesoscopic analysis of motion and conformation of cross-bridges

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 10¹²–10⁹ myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.     

Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.     

Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.     

The mechanism of muscle contraction

Knowledge of the mechanism of contraction has been obtained from studies of the interaction of actin and myosin in solution, from an elucidation of the structure of muscle fibers, and from measurements of the mechanics and energetics of fiber contraction. Many of the states and the transition rates between them have been established for the hydrolysis of ATP by actin and myosin subfragments in solution. A major goal is to now understand how the kinetics of this interaction are altered when it occurs in the organized array of the myofibril. Early work on the structure of muscle suggested that changes in the orientation of myosin cross-bridges were responsible for the generation of force. More recently, fluorescent and paramagnetic probes attached to the cross-bridges have suggested that at least some domains of the cross-bridges do not change orientation during force generation. A number of properties of active cross-bridges have been defined by measurements of steady state contractions of fibers and by the transients which follow step changes in fiber length or tension. Taken together these studies have provided firm evidence that force is generated by a cyclic interaction in which a myosin cross-bridge attaches to actin, exerts force through a "powerstroke" of 12 nm, and is then released by the binding of ATP. The mechanism of this interaction at the molecular level remains unknown.     

Structural characterization of weakly attached cross-bridges in the A*M*ATP state in permeabilized rabbit psoas muscle

It is well established that in a skeletal muscle under relaxing conditions, cross-bridges exist in a mixture of four weak binding states in equilibrium (A*M*ATP, A*M*ADP*P(i), M*ATP, and M*ADP*P(i)). It has been shown that these four weak binding states are in the pathway to force generation. In the past their structural, biochemical, and mechanical properties have been characterized as a group. However, it was shown that the myosin heads in the M*ATP state exhibited a disordered distribution along the thick filament, while in the M*ADP*P(i) state they were well ordered. It follows that the structures of the weakly attached states of A*M*ATP and A*M*ADP*P(i) could well be different. Individual structures of the two attached states could not be assigned because protocol for isolating the two states has not been available until recently. In the present study, muscle fibers are reacted with N-phenylmaleimide such that ATP hydrolysis is inhibited, i.e., the cross-bridge population under relaxing conditions is distributed only between the two states of M*ATP and A*M*ATP. Two-dimensional x-ray diffraction was applied to determine the structural characteristics of the attached A*M*ATP state. Because the detached state of M*ATP is disordered and does not contribute to layer line intensities, changes as a result of increasing attachment in the A*M*ATP state are attributable to that state alone. The equilibrium toward the attached state was achieved by lowering the ionic strength. The results show that upon attachment, both the myosin and the first actin associated layer lines increased intensities, while the sixth actin layer line was not significantly affected. However, the intensities remain weak despite substantial attachment. The results, together with modeling (see J. Gu, S. Xu and L. C. Yu, 2002, Biophys. J. 82:2123-2133), suggest that there is a wide range of orientation of the attached A*M*ATP cross-bridges while the myosin heads maintain some degree of helical distribution on the thick filament, suggesting a high degree of flexibility in the actomyosin complex. Furthermore, the lack of sensitivity of the sixth actin layer line suggests that the binding site on actin differs from the putative site for rigor binding. The significance of the flexibility in the A*M*ATP complex in the process of force generation is discussed.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.     

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.     

ATP induces microsecond rotational motions of myosin heads crosslinked to actin.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to study the effect of ATP on the rotational dynamics of spin-labeled myosin heads crosslinked to actin (XLAS1). We have previously shown that ATP induces microsecond rotational motions in activated myofibrils or muscle fibers, but the possibility remained that the motion occurred only in the detached phase of the cross-bridge cycle. The addition of ATP to the crosslinked preparation has been shown to be a model system for active cross-bridges, presumably providing an opportunity to measure the motion in the attached state, without interference from unattached heads. In the absence of ATP, XLAS1 had very little microsecond rotational mobility, yielding a spectrum identical to that observed for uncrosslinked acto-S1. This suggests that all of the labeled S1 forms normal rigor complexes when crosslinked to actin. The addition of 5 mM ATP greatly increased the microsecond rotational mobility of XLAS1, and the effects were reversed upon depletion of ATP. The most plausible explanation for these results is that myosin heads undergo microsecond rotational motion while attached actively to actin during steady state ATPase activity. These results have important implications for the interpretation of spectroscopic data obtained during muscle contraction.     

Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers.

The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AM*ADP.Pi state (A = actin, M = myosin). Since the state which leads into the AM*ADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AM*ADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AM*ADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.