Direct observation of molecular motility by light microscopy

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.     

The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Structure of the myosin projections on native thick filaments from vertebrate skeletal muscle

Rabbit psoas muscle filaments, isolated in relaxing buffer from non-glycerinated muscle, have been applied to hydrophilic carbon films and stained with uranyl acetate. Electron micrographs were obtained under low-dose conditions to minimize specimen damage. Surrounding the filament backbone, except in the bare zone, is a fringe of clearly identifiable myosin heads. Frequently, both heads of individual myosin molecules are seen, and sometimes a section of the tail can be seen connecting the heads to the backbone. About half the expected number of heads can be counted, and they are uniformly distributed along the filament. The majority of heads appear curved. The remainder could be curved heads viewed from another aspect. Three times as many heads curve in a clockwise sense than in an anticlockwise sense, suggesting a preferential binding of one side of the head to the carbon film. The two heads of myosin molecules exhibit all the possible combinations of clockwise, anticlockwise and straight heads, and analysis of their relative frequencies suggests that the heads rotate freely and independently. The heads also adopt a wide range of angles of attachment to the tail. The lengths of heads cover a range of 14 to 26 nm, with a peak at 19 nm. The average maximum width is 6.5 nm. Both measurements are in excellent agreement with values for shadowed molecules. Since our data are from heads adsorbed to the film in relaxing conditions and the shadowed molecules were free of nucleotide, gross shape changes are not likely to be produced by nucleotide binding. The length of the link between the heads and the backbone was found to vary between 10 nm and 52 nm, with a broad peak at about 25 nm. Thus, the hinge point detected in the tail of isolated molecules was not usually the point from which the crossbridges swung out from the filament surface. The angle made by the link to the filament axis was between 20 degrees and 80 degrees, with a broad maximum around 45 degrees. These lengths and angles concur with our observation of an average limit of the crossbridges from the filament surface of 30 nm. This is sufficient to enable heads in the myofibril lattice to reach out beyond the nearest thin filament and should allow considerable flexibility for stereospecific binding to actin in active muscle.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.     

Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.     

Myosin V exhibits a high duty cycle and large unitary displacement.

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.     

Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model

Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles,Revealed by Synchrotron X-Ray Fiber Diffraction

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.     

Structural changes of actin-bound myosin heads after a quick length change in frog skeletal muscle

Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

Packing of α-Helical Coiled-Coil Myosin Rods in Vertebrate Muscle Thick Filaments

Muscle myosin filament backbones are aggregates of long coiled-coil α-helical myosin rods, with the myosin heads arranged approximately helically on the filament surface, but the details of the rod packing are not known. Computed Fourier transforms of plausible molecular packing models for the vertebrate striated muscle myosin filament have been compared with observed high-angle X-ray diffraction patterns from plaice fin muscle. Models considered include those in which the coiled-coil rod parts of myosin are packed into various kinds of subfilaments or into a curved molecular crystalline layer. A general conclusion is that if the myosin rods are tilted by less than about 1° or more than about 3° from the filament long axis, very poor agreement is obtained between the computed and observed high-angle diffraction patterns. Qualitative comparison of calculated Fourier transforms, taken together with electron micrograph information, shows that the curved molecular crystal model and a model with hexagonally close-packed 4-nm subfilaments appear to explain the whole set of observations more satisfactorily than the alternatives. It is argued on other grounds that of these two possibilities the curved molecular crystal model is the more plausible.     

Ca2+ causes release of myosin heads from the thick filament surface on the milliseconds time scale

We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca2+. Negative staining reveals that when Ca2+ binds to the heads of relaxed Ca2+ -regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca2+. The structural change is thus a result of Ca2+ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca2+ -binding. We therefore used a time-resolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca2+ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca2+ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction.     

The myosin filament. VI. Myosin content.

There has been some disagreement about the number of myosin molecules in vertebrate skeletal myosin filaments calculated from the myosin to actin weight ratio determined by quantitative sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Tregear &; Squire, 1973; Potter, 1974; Morimoto &; Harrington, 1974). In this work it was found that (1) thoroughly washed fibrils are required to obtain the true value for the myosin to actin weight ratio. (2) Neither actin nor myosin is extracted preferentially during the required washing procedure. (3) There are four myosin molecules per 14.3 nm interval along the myosin filament or about 400 myosin molecules per filament.From published estimates of the number of molecules of C-protein per myosin filament (Offer et al., 1973; Morimoto &; Harrington, 1974) and the findings in this work, we conclude that there are four molecules of C-protein at each of the 14 C-protein binding positions along the filament, i.e. one C-protein molecule for each of the four myosin molecules contributing to the cross-bridges at each position.     

Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle

Thick filaments from leg muscle of tarantula, maintained under relaxing conditions (Mg-ATP and EGTA), were negatively stained and photographed with minimal electron dose. Particles were selected for three-dimensional image reconstruction by general visual appearance and by the strength and symmetry of their optical diffraction patterns, the best of which extend to spacings of 1/5 nm-1. The helical symmetry is such that, on a given layer-line, Bessel function contributions of different orders start to overlap at fairly low resolution and must therefore be separated computationally by combining data from different views. Independent reconstructions agree well and show more detail than previous reconstructions of thick filaments from Limulus and scallop. The strongest feature is a set of four long-pitch right-handed helical ridges (pitch 4 X 43.5 nm) formed by the elongated myosin heads. The long-pitch helices are modulated to give ridges with an axial spacing of 14.5 nm, lying in planes roughly normal to the filament axis and running circumferentially. We suggest that the latter may be formed by the stacking of a subfragment 1 (S1) head from one myosin molecule on an S1 from an axially neighbouring molecule. Internal features in the map indicate an approximate local twofold axis relating the putative heads within a molecule. The heads appear to point in opposite directions along the filament axis and are located very close to the filament backbone. Thus, for the first time, the two heads of the myosin molecule appear to have been visualized in a native thick filament under relaxing conditions.     

Application of potential energy calculations to the determination of muscle structure from X-ray data with special reference to the configuration of myosin heads

A method that relates molecular structure to the forces that maintain it and to its X-ray diffraction pattern is described and applied to muscle. In a computer model, the potential energy of the moveable components (here the myosin heads) is minimized by letting them move down the steepest gradient in three dimensions from a variety of starting positions. Initial values are assumed for the parameters that determine the forces, and for those that define the structure and arrangement of the fixed components. The X-ray pattern expected from the resulting structures can be calculated in a straightforward manner and compared with relevant observed data. Discrepancies can then be minimized by varying the values initially assumed for the parameters, as in the conventional “trial and error” method.This first application of the present method is concerned with the effects of the hexagonal lattice on the myosin head configuration in thick filaments of the type found in vertebrate skeletal muscle. For that purpose, a very simple model was used with the following main features: smooth cylinders for the thin filaments and for the thick filament backbones, two spherical heads attached by Hookean springs to each point of a $\text{9}\text{3}$ helix on the surface of the backbone, and repulsive forces of the electrostatic double-layer type acting between each head and all other surfaces.The myosin head configuration was calculated for an isolated thick filament and a study was made of the effects of packing such filaments into a hexagonal lattice of various side spacings in the presence or absence of thin filaments. For the isolated filament, it was found that the $\text{9}\text{3}$ helical symmetry is maintained in the myosin head configuration and that the two heads of each molecule are splayed azimuthally. When such filaments are packed into the hexagonal lattice with thin filaments present, the $\text{9}\text{3}$ helical symmetry of the myosin head configuration is lost. As the lattice side spacing is reduced, the myosin heads become increasingly displaced not only in the radial and azimuthal directions but also in the axial direction, although they interact primarily with smooth cylinders. The axial separation of the two heads in each molecule becomes different in one level from that in the other two in the 43 nm axial repeat, thus increasing the repeat in projection onto the axis from 14.3 to 43 nm. This effect may contribute to the “forbidden meridionals” described by Huxley & Brown (1967). In the absence of thin filaments, the displacements of the myosin heads are much smaller, even when the lattice side spacing is reduced to that present in muscles stretched to non-overlap.Applying the method based on potential energy minimization to the evaluation of X-ray data from muscles in hypertonic Ringer reveals that, even in the case of patterns apparently free of lattice sampling (and thus normally considered to represent diffraction from single filaments), the interpretation must include the nearest myosin heads from neighbouring filaments, and that this may be necessary also for unsampled patterns obtained from muscles in normal Ringer. Furthermore, the method helps to explain several other major features of X-ray results obtained from muscles in the hypertonic state and from muscles stretched in normal Ringer to long sarcomere lengths including non-overlap. It is concluded that the method provides a powerful tool for the interpretation of muscle X-ray patterns.     

Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This “interacting-heads motif” (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3–4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3–4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.     

Distribution of mass within native thick filaments of vertebrate skeletal muscle

The distribution of mass within the vertebrate skeletal thick filament has been determined by scanning transmission electron microscopy. Thick and thin filaments from fresh rabbit muscle were mixed with tobacco mosaic virus (TMV), fixed with formaldehyde, dried onto thin carbon films and viewed in a computer-linked microscope. Electron scattering data from both TMV and thick filaments were analysed with reference to the long axis of the particles so that the distribution of mass within the particles could be determined. While TMV appeared to be a uniform rod at the resolution employed (4.3 nm), the thick filament was clearly differentiated along its length. M-line remnants at the centre of the filament were flanked by regions of low mass per unit length, corresponding to the bare zone of the filament, and then by the more massive cross-bridge regions. The mass per unit length was approximately constant through most of the cross-bridge zone and declined at the filament tips, in a manner consistent with a constant number of myosin molecules per 14.3 nm interval (crown) throughout the cross-bridge zone. Fourier analysis of the data failed to detect the expected 43 nm periodicity of C-protein. The total mass of the thick filament was 184 Mdalton (s.e.m., 1.6 X 10(6); n = 70). The mass of adhering M-line proteins was highly variable but, on average, was about 4 Mdalton. The total mass of the filament and the mass distribution in the cross-bridge zone are consistent with three myosin molecules per crown.     

Effect of pH on the cross-bridge arrangement in synthetic myosin filaments.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.