丝瓜肌球蛋白的电子显微学研究

从丝瓜(Luffa cylindrica (L.) Roem.)卷须中纯化得到分子量为174kD的肌球蛋白,并对其进行了酶学与电子显微学的研究.这种肌球蛋白具有肌动蛋白激活的MgATPase活性,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别.电子显微学研究表明:它有两个头部(大小和形状与动物肌肉的肌球蛋白相似)和一条相对较短的尾部.还对丝瓜卷须的肌动蛋白进行了观测,偶尔发现一些尾部有球状结构的肌球蛋白.该肌球蛋白的免疫特性和超微结构证明了它由2条重链组成,并与传统的肌球蛋白相似.然而,这种174 kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究.     

Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin

It has been shown that skeletal and smooth muscle myosin heads binding to actin results in the movement of smooth muscle tropomyosin, as revealed by a change in fluorescence resonance energy transfer between a fluorescence donor on tropomyosin and an acceptor on actin (Graceffa, P. (1999) Biochemistry 38, 11984-11992). In this work, tropomyosin movement was similarly monitored as a function of unphosphorylated and phosphorylated smooth muscle myosin double-headed fragment smHMM. In the absence of nucleotide and at low myosin head/actin ratios, only phosphorylated heads induced a change in energy transfer. In the presence of ADP, the effect of head phosphorylation was even more dramatic, in that at all levels of myosin head/actin, phosphorylation was necessary to affect energy transfer. It is proposed that the regulation of tropomyosin position on actin by phosphorylation of myosin heads plays a key role in the regulation of smooth muscle contraction. In contrast, actin-bound caldesmon was not moved by myosin heads at low head/actin ratios, as uncovered by fluorescence resonance energy transfer and disulfide cross-linking between caldesmon and actin. At higher head concentration caldesmon was dissociated from actin, consistent with the multiple binding model for the binding of caldesmon and myosin heads to actin (Chen, Y., and Chalovich, J. M. (1992) Biophys. J. 63, 1063-1070).     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

A new model of cooperative myosin-thin filament binding

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.     

Actomyosin adenosine triphosphatase regulation by intramolecular myosin mechanisms. Myosin light chains functions and rod modification effects

Mechanisms of the actomyosin ATPase modulation via the myosin light chains (LC) in various myosin types are discussed. The essential LC increase the stability of the myosin heavy chains (HC) in the myosin heads and, under certain conditions, they can affect the degree of interaction of HC with actin. The regulatory LC (RLC) are sensitive to calcium binding on specific sites or to calcium activated phosphorylation. These factors induce changes of the RLC state followed by changes of the HC state in response to calcium concentration changes during the contractile process. Direct calcium binding or phosphorylation effects in various muscles are mediated by special types of RLC and HC. Several examples of actomyosin ATPase changes induced by modifications of the myosin rod are compared. A common feature of these effects is a possible involvement of certain configurational changes of the myosin molecule. These changes can affect the spatial position of the myosin heads and the myosin-actin interaction.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Regulatory light-chain Cys-55 sites on the two heads of myosin can come within 2A of each other.

The di-thiol reagent, 5,5'-dithiobis (2-nitrobenzoic acid) is shown to induce disulfide bond formation between Mercenaria regulatory light-chain Cys-55 sites on either head of scallop hybrid myosin. This indicates that these two sites on opposite heads of myosin can come within 2A of each other and this confirms a prediction based on earlier data [Chantler, Tao and Stafford (1991) Biophys. J. 59, 1242-1250]. Results demonstrate that myosin heads in solution show a considerable mutual freedom of movement which can be monitored by probes in the vicinity of regulatory light-chain residue 55. Implications for light-chain movement on the myosin head are discussed.     

A deterministic mechanism producing the loose coupling phenomenon observed in an actomyosin system

Myosins are molecular motors that convert the chemical energy of ATP into mechanical work called a power stroke. Class II myosin engaged in muscle contraction is reported to show a "loose coupling phenomenon", in which the number of power strokes is greater than the number of ATP hydrolyses. This phenomenon cannot be explained by the lever-arm hypothesis, which is currently accepted as a standard theory for myosin motility. In this paper, a model is proposed to reproduce the loose coupling phenomenon. The model is based on a mechanochemical process called "Driven by Detachment (DbD)" mechanism, which assumes that the energy of the power strokes originates from the potential energy generated by the attractive force between myosin and actin. During the docking process, the potential energy is converted into an intramolecular strain in a myosin molecule, which drives the power stroke after the myosin is firmly attached to an actin filament. The energy of ATP is used to temporarily reduce the attractive force and to increase the potential energy. Therefore, it is not directly linked to the power strokes. When myosin molecules work as an aggregate, the sliding movement of a myosin filament driven by the power strokes of some myosin heads makes other myosin heads that have completed their power strokes detach from the actin without consuming ATP. Under the DbD mechanism, these passively detached myosins can be again engaged in power strokes after the next attachment to actin. As a result, the number of power strokes becomes greater than the number of ATP hydrolyses, and the loose coupling phenomenon will be observed. A theoretical analysis indicates that the efficiency of converting the potential energy into intramolecular elastic energy determines the number of power strokes per each ATP hydrolysis. Computer simulations showed that the DbD mechanism actually produced the loose coupling phenomenon. A critical requirement for this mechanism is that ATP must preferentially facilitate the detachment of myosins that have completed their power strokes, but are still strongly attached to the actin. This requirement may be fulfilled by ATP hydrolysis tightly depending on the conformation of a myosin molecule.     

Diffuse X-ray scatter from myosin heads in oriented synthetic filaments.

X-ray results are presented concerning the structural state of myosin heads of synthetic filaments in threads. These were made from purified rabbit skeletal muscle myosin and studied by x-ray diffraction and electron microscopy by Cooke et al. (Cooke, P. H., E. M. Bartels, G. F. Elliott, and R. A. Hughes, 1987, Biophys. J., 51:947-957). X-ray patterns show a meridional peak at a spacing of 14.4 nm. We concentrate here on the only other feature of the axial pattern: this is a central region of diffuse scatter, which we find to be similar to that obtained from myosin heads in solution (Mendelson, R. A., K. M. Kretzschmar, 1980, Biochemistry, 19:4103-4108). This means that the myosin heads have very large random displacements in all directions from their average positions, and that they are practically randomly oriented. The myosin heads do not contribute to the 14.4-nm peak, which must come entirely from the backbone. Comparison with x-ray data from the unstriated Taenia coli muscle of the guinea pig indicates that in this muscle at least 75% of the diffuse scatter comes from disordered myosin heads. The results confirm that the diffuse scatter in x-ray patterns from specimens that contain myosin filaments can yield information about the structural behavior of the myosin heads.     

A new model for the surface arrangement of myosin molecules in tarantula thick filaments

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head.We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing.Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.     

The force exerted by a muscle cross-bridge depends directly on the strength of the actomyosin bond

Myosin produces force in a cyclic interaction, which involves alternate tight binding to actin and to ATP. We have investigated the energetics associated with force production by measuring the force generated by skinned muscle fibers as the strength of the actomyosin bond is changed. We varied the strength of the actomyosin bond by addition of a polymer that promotes protein-protein association or by changing temperature or ionic strength. We estimated the free energy available to generate force by measuring isometric tension, as the free energy of the states that precede the working stroke are lowered with increasing phosphate. We found that the free energy available to generate force and the force per attached cross-bridge at low [Pi] were both proportional to the free energy available from the formation of the actomyosin bond. We conclude that the formation of the actomyosin bond is involved in providing the free energy driving the production of isometric tension and mechanical work. Because the binding of myosin to actin is an endothermic, entropically driven reaction, work must be performed by a "thermal ratchet" in which a thermal fluctuation in Brownian motion is captured by formation of the actomyosin bond.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Effect of activation by calcium on the x-ray diffraction pattern from insect flight muscle.

A low-angle X-ray diffraction pattern of calcium-activated Lethocerus flight muscle was formed and the intensities of various parts of the pattern observed by means of a proportional counter. The muscle was sinusoidally oscillated in length to produce mechanical work. The resultant changes in diffraction intensity were related to the state of the muscle and to the phase of the mechanical oscillatory cycle. The measurements were interpreted in terms of a movement of the heads of the myosin molecules into contact with the actin filaments. In these terms the results showed that between 10 and 20% of the myosin heads attached to actin during work-producing oscillation of the muscle. The time-course of this attachment followed that of tension generation with a small delay. Calculation suggests that not all of the myosin molecules attached to actin at any one moment were generating tension.     

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.     

A model to account for the elastic element in muscle crossbridges in terms of a bending myosin rod

We advance a structural model to account for the rapid elastic element seen in mechanical transient experiments on vertebrate skeletal muscle (A.F. Huxley & Simmons 1971 Nature, Lond. 233, 533-538). In contrast to other crossbridge models, ours does not envisage a myosin rod made up of two rigid portions connected by a hinge, but rather a gradually bending rod portion connecting the heads to the thick filament shaft. We propose that, in relaxed muscle, the subfragment 2 (S2) portion of the myosin rod is bound to the thick filament shaft by ionic interactions analogous to those between the light meromyosin (LMM) portions of the rod that constitute the body of the shaft. These interactions probably involve the alternating zones of positive and negative charge seen in myosin rod amino acid sequences. As the crossbridge cycle that generates tension begins, we propose that part of S2 detaches from the thick filament shaft and bends to enable the myosin head to attach to actin. When tension develops in the crossbridge, the S2 is straightened and more of it becomes detached from the shaft so that the junction between S2 and the myosin heads moves 3-4 nm axially. As tension declines at the end of the crossbridge stroke, we propose that S2 rebinds to the thick filament shaft and that this provides the restoring force to return the junction of the heads and S2 to its original axial position. Thus this movement would have the characteristics of an elastic element; detailed calculations indicate that it would have properties similar to those observed experimentally. Furthermore, this model can account for the radial attractive force seen in rigor and in contracting muscle, the decrease in stiffness when interfilament spacing is increased in skinned muscle, and the increased rate of proteolysis observed at the S2-LMM junction in contracting muscle.     

Emergent Systems Energy Laws for Predicting Myosin Ensemble Processivity

In complex systems with stochastic components, systems laws often emerge that describe higher level behavior regardless of lower level component configurations. In this paper, emergent laws for describing mechanochemical systems are investigated for processive myosin-actin motility systems. On the basis of prior experimental evidence that longer processive lifetimes are enabled by larger myosin ensembles, it is hypothesized that emergent scaling laws could coincide with myosin-actin contact probability or system energy consumption. Because processivity is difficult to predict analytically and measure experimentally, agent-based computational techniques are developed to simulate processive myosin ensembles and produce novel processive lifetime measurements. It is demonstrated that only systems energy relationships hold regardless of isoform configurations or ensemble size, and a unified expression for predicting processive lifetime is revealed. The finding of such laws provides insight for how patterns emerge in stochastic mechanochemical systems, while also informing understanding and engineering of complex biological systems.     

"Crystalline" myosin cross-bridge array in relaxed bony fish muscle. Low-angle x-ray diffraction from plaice fin muscle and its interpretation

Detailed structural analysis of muscles normally used to study myosin cross-bridge behavior (e.g., frog sartorius muscle, insect flight muscle) is extremely difficult due to the statistical disorder inherent in their myosin filament arrays. Bony fish muscle is different from all other muscle types in having a myosin filament (A-Band) array with good three-dimensional (crystalline) regularity that is coherent right across each myofibril. Rigorous structure analysis is feasible with fish muscle. We show that low-angle x-ray diffraction patterns from plaice fin muscle contain characteristic vertebrate layer lines at orders of 429 (+/- 0.2) A, that these layer lines are well sampled by row-lines from a simple hexagonal lattice of a-spacing 470 (+/- 2.0) A at rest length and that there are meridional reflections, due to axial perturbations of the basic helix of myosin heads, similar in position to those from frog muscle but differing in relative intensities. Clear trends based on modeling to a resolution of 130 A of the observed intensities in the low angle x-ray diffraction pattern from relaxed plaice fin muscle suggest that: (a) the pattern out to 130 A is more sensitive to the distribution of the two heads than it is to details of the head shape, (b) both heads in one myosin molecule probably tilt axially in the same direction by approximately 20-40 degrees relative to a normal to the thick filament backbone, (c) the center of mass of the heads is at 145 to 160 A radius, and (d) the two heads form a compact structure by lying closely adjacent to each other and almost parallel. Little rotational disorder of the heads can occur. Because of its crystallinity, bony fish muscle provides a uniquely useful structural probe of myosin cross-bridge behavior in other muscle states such as rigor and active contraction.     

A physical model of ATP-induced actin-myosin movement in vitro.

The nature of the mechanism limiting the velocity of ATP-induced unidirectional movements of actin-myosin filaments in vitro is considered. In the sliding process two types of "cyclic" interactions between myosin heads and actin are involved, i.e., productive and nonproductive. In the productive interaction, myosin heads split ATP and generate a force which produces sliding between actin and myosin. In the nonproductive interaction "cycle," on the other hand, myosin heads rapidly attach to and detach from actin "reversibly," i.e., without splitting ATP or generating an active force. Such a nonproductive interaction "cycle" causes irreversible dissipation of sliding energy into heat, because the myosin cross-bridges during this interaction are passive elastic structures. This consideration has led us to postulate that such cross-bridges, in effect, exert viscous-like frictional drag on moving elements. Energetic considerations suggest that this frictional drag is much greater than the hydrodynamic viscous drag. We present a model in which the sliding velocity is limited by the balance between the force generated by myosin cross-bridges in the productive interaction and the frictional drag exerted by other myosin cross-bridges in the nonproductive interaction. The model is consistent with experimental findings of in vitro sliding, including the dependence of velocity on ATP concentration, as well as the sliding velocity of co-polymers of skeletal muscle myosin and phosphorylated and unphosphorylated smooth muscle myosins.     

Evidence for cooperative interactions of myosin heads with thin filament in the force generation of vertebrate skeletal muscle fibers

To examine the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA-and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-phenylenedimaleimide (p-PDM)-treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one, and two of their heads inactivated, and the relationships among these values (expressed relative to the control values) were studied. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, muscle fiber stiffness is determined by the total number of native heads, and there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.     

Why Muscle is an Efficient Shock Absorber

Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.