Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.     

Hydrostatic pressure studies of native and synthetic thick filaments: in vitro myosin aggregates at pH 7.0 with and without C-protein

Column-purified myosin at pH 7.0 will reproducibly aggregate into filaments of known average length and structure when dialyzed against a low ionic strength medium under controlled conditions. When exposed to increased hydrostatic pressure, followed by quick return to atmospheric pressure, the original filaments shorten linearly with increasing pressure; in addition, a second population of filaments is seen, presumably the result of reaggregation of myosin after release of pressure. This second population is about 0.5 microns long, bipolar, and about half the diameter of the original filaments. The number of these filaments, but not their physical characteristics, is a function of the shortening of the original filament population. Both the remnants of the original population and the new aggregates, once formed, are stable over time and at room temperature. The addition of C-protein to myosin solutions before filament preparation results in a filament population of slightly shorter length. When these filaments are exposed to increased hydrostatic pressure, they are more resistant to disaggregation than myosin filaments without C-protein. However, like the filaments prepared in the absence of C-protein, a second population of shorter, thinner filaments is visible after exposure to pressure.     

Electron microscope studies of thick filaments from vertebrate skeletal muscle.

Separated thick filaments have been prepared for electron microscopy by a method involving freeze-drying and shadowing. In the resulting filaments the individual heads of myosin molecules can be seen surrounding the filament shaft, which appears relatively smooth. Pairs of heads can frequently be seen to be emanating from a common origin. Myosin heads are found at distances up to 500 Å from the edge of the shaft.     

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.     

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.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle.

1. The molecular weights of a series of synthetic myosin filaments have been measured, using the transport-concentration dependence theory of Rowe, A.J. [Biopolymers, 1977, 16, 2595--2611]. It is shown that for preparations of narrow length distribution (0.60--0.77 micrometer), N, the number of myosin molecules/14.3 nm varies between 3 and 6. 2. The reduced specific viscosity of synthetic myosin filaments has been measured as a function of both concentration and shear rate. From the concentration dependence at zero rate of shear, a value for the "swelling" of the filaments Vs/-v = 2.3 has been calculated. 3. The frictional coefficient of synthetic myosin filaments has been shown to be anomalously but reproducibly high, as compared to that of prolate ellipsoids of the same length and mass. This additional frictional drag has been numerically characterised by a "frictional increment", fi = 1.76 +/- 0.11. 4. A procedure has been devised whereby for any elongated structure which can be assumed to show the same (or other known) fi value, the molecular weight can be estimated from s0 (extrapolated sedimentation coefficient) and 2b (length) alone. 5. An s0 value for natural A-filaments, isolated from rabbit psoas muscle, has been determined by the active enzyme centrifugation technique. From this value, s0 = 132 +/- 3 S, a molecular weight of 1.20 . 10(8) has been computed by the new procedure, for preparations of average length 1.27 micrometer. 6. Contingent upon the validity of the assumptions used (see 4 above) the N value is computed as 3.1 +/- 0.2, consistent with the native, fully intact A-filament having three-fold symmetry, containing 294 myosin molecules, and having a molecular weight based upon myosin and C-protein of 1.31 . 10(8).     

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.     

Hydrostatic pressure rescues native protein from aggregates.

Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods.     

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.     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

End-filaments: A new structural element of vertebrate skeletal muscle thick filaments

The use of low ionic strength buffers to dissociate separated thick filaments into three subfilaments is described. When the dissociation is performed in solution, rather than on an electron microscope grid, structures called end-filaments are observed where the subfilaments terminate. The end-filaments, only one of which is seen for every three subfilaments, are about 850 Å long, 50 Å wide and show transverse striations with a periodicity of 42 Å.     

Characterization of sarcolemma from rabbit skeletal muscle: developmental studies

Sarcolemmal membranes were obtained from skeletal muscle of rabbits at different developmental stages. Lipid and protein composition, as well as enzymatic activities, were compared in sarcolemma prepared in vesicular form. During the developmental period no distinct changes in lipid content and composition were found. Some changes in protein pattern and activities of marker enzymes (5′-nucleotidase and ouabain-sensitive Na⁺,K⁺-ATPase) were detected. Also, changes in Mg²⁺-ATPase and Mg²⁺, Ca²⁺-ATPase activities during development were found.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle

With a view to obtaining a more complete view of the composition and structure of the thick filaments of vertebrate skeletal muscle, we have isolated and characterized two new myofibrillar components, H-protein and X-protein. These were purified by hydroxyapatite column chromatography of an impure C-protein preparation itself made from impure myosin extracted from rabbit back and leg muscles. H-protein is the protein responsible for band H on sodium dodecyl sulphate/polyacrylamide gel electrophoresis of crude myosin. X-protein, although present in such preparations in significant quantities, was not detected previously since it is difficult to resolve from C-protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Physical-chemical parameters have been determined for the new proteins and compared with those of C-protein. The apparent chain weight of H-protein estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis is 69,000, whereas that of X-protein (152,000) is only slightly greater than that of C-protein (140,000). The molecular weights of H- and X-proteins determined by sedimentation equilibrium centrifugation show that the molecules contain only a single polypeptide chain. The circular dichroism spectra indicate that the proteins have low alpha-helical contents. Both proteins, particularly H-protein, have a high proline content. Although X-protein is of similar chain weight to C-protein, the two show distinct differences in other properties. The sedimentation coefficient of X-protein is markedly lower than that of C-protein, suggesting X-protein is a more asymmetrical molecule. The amino acid compositions, although broadly similar, also show clear differences. Antibodies to H-protein, X-protein and C-protein have been raised in goats and shown not to cross-react.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.     

Structure of short thick filaments from Limulus muscle

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.     

Thin filaments of rabbit skeletal muscle are in helical register

Thin sections of rapidly frozen and freeze-substituted rabbit glycerinated muscle fibres loaded with myosin subfragment-1 were used to examine a three-dimensional arrangement of thin filaments in vertebrate skeletal muscle. Clearer images of the "arrowhead" structure were obtained when specimens were freeze-substituted first in a tannic acid solution and then in an OsO4 solution. The images obtained showed that the arrowheads were aligned laterally. This indicates that all the thin filaments have the same rotational orientation in a half sarcomere of rabbit skeletal muscle in the rigor state.     

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.