The structure of spindle-shaped paracrystals of light meromyosin

Light meromyosin paracrystals have been studied by electron microscopy combined with optical diffraction in order to understand how the tails of the myosin molecules might pack in the backbone of muscle thick filaments. The forms of paracrystal investigated were all spindle-shaped structures with an axial periodicity of either 43 nm or 14.3 nm or hybrids involving aspects of both repeats. Transverse sections show that they are not smooth but polygonal in outline. Analysis of the band patterns in negatively stained specimens indicates that the molecular arrangement in the paracrystals involves both parallel and antiparallel interactions. A parallel axial displacement of the molecules by 43 nm is intrinsic to all forms of paracrystal investigated. The principal antiparallel overlap between molecules appears to be one of 84 nm, and it is suggested that an antiparallel dimer is the structural unit in the paracrystals. The role of the interactions leading to these displacements in the formation of the thick filament backbone is discussed.     

The axial repeats in paracrystals of light meromyosin and its complex with C-protein

We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.     

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.     

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.     

The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

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.     

Effects of light chain phosphorylation and skeletal myosin on the stability of non-muscle myosin filaments

The effect of light chain phosphorylation and the presence of skeletal muscle myosin on the stability of non-phosphorylated non-muscle myosin filaments was investigated. Purified skeletal, brush border and thymus myosins were assembled in vitro into hybrid filaments consisting of varying proportions of (1) non-muscle and skeletal myosins, or (2) phosphorylated and non-phosphorylated non-muscle myosins. The stability of these hetero- and homopolymers in the presence of MgATP was determined using sedimentation, gel electrophoresis and immunochemical techniques. In addition, the effect of a monoclonal antibody, binding to the tip of brush border myosin tail, on the assembly of the homo- and heteropolymers, was tested. Filamentous non-phosphorylated non-muscle myosin was disassembled by MgATP to the same extent whether in homo- or heteropolymers, indicating that skeletal myosin has no stabilising effect on the hybrid filaments. The presence of small amounts of phosphorylated non-muscle myosin was, however, found to prevent the complete disassembly by MgATP of non-phosphorylated non-muscle myosin filaments, indicating that light chain phosphorylation stabilizes co-operatively non-muscle myosin filaments. The monoclonal antibody prevented the assembly of brush border myosin into both homo- and heteropolymers, and its effect on the filaments was compared with that of MgATP.     

Comparative studies of light meromyosin paracrystals derived from red, white, and cardiac muscle myosins

Tryptic and chymotryptic light meromyosin paracrystals from red and cardiac muscles of rabbit show a negative and positive staining pattern with uranyl acetate and phosphotungstate that sharply differs from that of white muscle light meromyosin paracrystals. The main periodicity of about 430 A is the same regardless of the source of light meromyosin. The results are discussed in terms of the molecular structure and the functional properties of various myosins.     

Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

Expression of light meromyosin in Dictyostelium blocks normal myosin II function

The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha- helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Thermal unfolding of myosin rod and light meromyosin: circular dichroism and tryptophan fluorescence studies

Rabbit skeletal myosin rod, which is the coiled-coil alpha-helical portion of myosin, contains two tryptophan residues located in the light meromyosin (LMM) portion whose fluorescence contributes 27% to the fluorescence of the entire myosin molecule. The temperature dependence of several fluorescence parameters (quantum yield, spectral position, polarization) of the rod and its LMM portion was compared to the thermal unfolding of the helix measured with circular dichroism. Rod unfolds with three major helix unfolding transitions: at 43, 47, and 53 degrees C, with the 43 and 53 degrees C transitions mainly located in the LMM region and the 47 degrees C transition mainly located in the subfragment 2 region. The fluorescence study showed that the 43 degrees C transition does not involve the tryptophan-containing region and that the 47 degrees C transition produces an intermediate with different fluorescence properties from both the completely helical and fully unfolded states. That is, although the fluorescence of the 47 degrees C intermediate is markedly quenched, the tryptophyl residues do not become appreciably exposed to solvent until the 53 degrees C transition. It is suggested that although the intermediate that is formed in the 47 degrees C transition contains an extensive region which is devoid of alpha-helix, the unfolded region is not appreciably solvated or flexible. It appears to have the properties of a collapsed nonhelical state rather than a classical random coil.     

Heavy meromyosin decoration of filaments fromEscherichia coli

A fibrous protein complex extracted fromEscherichia coli B/r by the method of Minkoff and Damadian [2] demonstrates arrowhead complexes when reacted with heavy meromyosin.