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.     

Dynamic light-scattering study of synthetic myosin filaments

Synthetic myosin filaments of rabbit were prepared. Electron microscopy showed that the number-average length (L_n = 470 nm) and sharpness in length distribution (L_w/L_n = 1.03₆) were independent of ionic strengths of 134, 74, and 44 mM, whereas the number ratio of M-filaments (about 15 nm in diameter at the bare zone) to m-filaments (about 10 nm) strongly depended on ionic strength (IS); the major filaments were M-filaments at IS = 134 mM, m-filaments at IS = 74 mM, and almost exclusively m-filaments at IS = 44 mM. Dynamic light scattering showed that the change in diameter with the change in ionic strength by 2-h dialysis was reversible. Combination of dynamic light scattering and sedimentation studies suggested a dynamic equilibrium between M- and m-filaments. Dynamic light-scattering spectra at IS = 134 and 74 mM could be analyzed by a theory for rigid rods, whereas those at IS = 44 mM only by introducing semiflexibility of filaments; m-filaments are more flexible at IS = 44 than at 74 mM.     

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.     

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).     

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.     

Interaction of aldolase with actin-containing filaments. Structural studies.

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

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.     

Electron microscopy of synthetic myosin filaments. Evidence for cross- bridge. Flexibility and copolymer formation

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.     

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.     

The actomyosin ATPase of synthetic myosin minifilaments, filaments, and heavy meromyosin

The actin-activated ATPase activities of myosin minifilaments and heavy meromyosin are similar at high actin concentrations. Under low ionic strength conditions, the minifilaments in Tris citrate buffer yield the same maximal turnover rate (Vmax) and apparent dissociation constant of actin from myosin (Kapp) as heavy meromyosin in standard low salt conditions. The time course of actin-activated ATP hydrolysis of minifilaments is similar to that observed for standard myosin preparations. Depending on the exact protein composition of the assay mixture, either the ATPase activity declines continuously with time, or is accelerated at the onset of superprecipitation. In analogy with myosin filaments, the ATPase of minifilaments shows a biphasic dependence on actin concentration. Super-precipitation of minifilaments follows a well resolved clearing phase during which their structural integrity appears to be fully preserved. These results indicate that minifilaments or similar small assemblies of myosin can fulfill contractile functions.     

Calorimetric studies of the ADP binding to myosin subfragment 1, heavy meromyosin, and to myosin filaments.

A calorimetric titration method was used to study the ADP binding to the chymotryptic subfragments of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1), and to myosin aggregated into filaments at low ionic strength. The binding constant (K) and heat of reaction (deltaH, kiloJoules (moles of ADP bound)-1) were determined. For HMM in 0.5 M KCl, 0.01 M MgCl2, 0.02 M Tris (pH 7.8) at 12 degrees, log K = 5.92 +/- 0.13 and deltaH = -70.9 +/- 3.6 kJ mol-1. These results agree with our previous findings for myosin in 0.5 M KCl at 12 degrees. When the KCl concentration was reduced to 0.1 M, the binding constant did not change significantly (log K = 6.09 +/- 0.06) but the binding was more exothermic (deltaH = -90.1 +/- 3.3 kJ mol-1). Similar results were obtained for myosin filaments in 0.1 M KCl and also for both the isoenzymes of S-1(S-1(A1) and S-1(A2) in 0.1 M KCl. In 0.5 M KCl, the binding curves suggest that about one ADP is bound per active site, but as 0.1 M KCl, the apparent stoichiometry drops from 0.7 to 0.75. The most probable explanation is that there is some site heterogeneity which is more evident at lower ionic strength.