Structural characterization of myosin from bovine brain

Myosins isolated from bovine brain, rabbit skeletal muscle, and chicken gizzard smooth muscle and their heavy meromyosin and light meromyosin fractions were studied in the electron microscope by negative staining with uranyl acetate. Under similar conditions of preparation and polymerization, the three myosins formed paracrystals of different structures. The light meromyosin portion of the skeletal muscle myosin also assembled in a different fashion than the brain or smooth muscle light meromyosins; the latter two assembled similarly. The heavy meromyosin portion from each of the three myosins was shown to interact with the actins isolated from each of the three tissue sources by the formation of the characteristic arrowhead patterns with similar periodicities. The brain heavy meromyosin attachment to both skeletal and brain actins was dissociated by ATP. It is suggested that differences in the light meromyosin portions of the three myosins may account in part for their differences in assembly in vivo.     

Molecular interactions in myosin assembly. Role of the 28-residue charge repeat in the rod.

We have used internal deletions of multiples of seven residues to change the phase of the 28-residue charge repeat in a light meromyosin cDNA construct expressed in Escherichia coli. The solubility behaviour of these mutants was similar to that of the wild-type material, but the molecular packing in the aggregates formed at low ionic strength was different. Whereas wild-type material formed paracrystals in which molecules were in close contact over most of their length, molecules in the paracrystals formed by the mutants were in close contact for only a short distance, which was just short enough to exclude the deletion from the overlap. These data indicate that, although the 28-residue charge periodicity is important in myosin molecular interactions, it is probably not the major driving force for myosin assembly and instead influences the detailed axial stagger of the interacting molecules.     

Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

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.     

SOME PROPERTIES OF EMBRYONIC MYOSIN

Myosins from the following sources were purified by diethylaminoethyl-Sephadex chromatography: moytubes grown in vitro for 7–8 days, prepared from pectoralis muscles of 10-day old embryos, and breast and leg muscles from 16-day old embryos. The adenosine triphosphatase activities of these myosins were close to that of adult m. pectoralis myosin. The light chains of the embryonic myosins had the same mobilities in sodium dodecyl sulfate electrophoresis as those in adult pectoralis muscle myosin and were clearly distinguishable from those in myosin from tonic muscle m. latissimus dorsi anterior. The fastest light chain in embryonic muscle myosin—apparent mol wt 16,000—was present in smaller amounts than in adult myosin. The negative staining pattern of paracrystals of embryonic light meromyosin (LMM) was indistinguishable from that of adult fast muscle LMM. The significance of these results for differentiation of various muscle types has been discussed.     

Determination of quantitative parameters of the binding of F-protein (phosphofructokinase) with myosin and localization of binding sites on the myosin molecule

To determine the localization of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM K-phosphate pH 6.5 was studied, using sedimentation, electron microscopic and optical diffraction methods. Sedimentation experiments showed that F-protein binds to myosin and myosin rod rather than to light meromyosin or S-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of F-protein binding sites on myosin calculated per chain weight of 80 000 is 5 +/- 1. The sedimentation results were confirmed by electron microscopic data. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added before or after filamentogenesis. A comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones revealed a marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case.     

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.     

Structural and functional changes of myosin during development: comparison with adult fast, slow and cardiac myosin.

ATPase (Ca²⁺ and K⁺ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO₄ polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC₂ except that the relative amount of LC₃ is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.     

Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.     

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.     

Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon. Effects of pH, salt, temperature, and troponin binding.

Tropomyosin (Tm) paracrystal formation induced by Mg2+ was studied by monitoring increases in light scattering. Paracrystals formed above a critical Tm concentration with lag phases in the time courses at pH 7.5 and 6.0, indicating that condensation polymerization processes are involved. The kinetic data at pH 7.5 reasonably fit a model in which nucleation and elongation are taken into account. The rate and extent of light scattering increased at low [Mg2+] and decreased at high [Mg2+] with a maximum at [Mg2+] = 15 mM, indicating different effects of Mg2+ in the two [Mg2+] ranges. The paracrystals were destabilized by increasing the salt concentration and decreasing the temperature. Mg2+ produces paracrystals at pH 6.0 and pH 7.5 by different kinetic mechanisms. Different Tm intermolecular interactions at the two pH values were indicated by studies of the excimer fluorescence of pyrene-labeled Tm and by effects of salt and temperature on the kinetics. At pH 6.0 Tm more readily formed paracrystals with decreased electrostatic effects. Effects of troponin on Mg2+-paracrystal formation of Tm at the two pH values correlated with the known differences in paracrystal structure when troponin is bound to Tm.     

Regions necessary for pH-dependent assembly of gizzard myosin rod

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod.     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail

Xenopus lamin A and a lamin A mutant lacking the complete 280 amino acid long carboxy-terminal tail were expressed in bacteria and purified from inclusion bodies. Electron microscopic analysis of lamin A dimers revealed that the carboxy-terminal 280 amino acids correspond to the globular domain seen in rotary-shadowed wild-type lamin and that the rodlike domain consists of the short non-helical amino terminus and the alpha-helical region. During reconstitution lamin A dimers first formed polar head to tail aggregates which then associated laterally resulting in paracrystals with periodic repeats of 25 nm. In the mutant, the longitudinal and lateral association of dimers had not been influenced, however, periodic repeats were absent in the filament bundles formed. Thus our data clearly demonstrate that carboxy-terminal tails are localized in light-stained regions of negatively stained paracrystals and that they are responsible for the alternating light dark staining of paracrystals. Fibrils, 2 to 3 nm thick, were a common structural element of paracrystals and filament bundles.     

Effects of actin and calcium ion on chymotryptic digestion of skeletal myosin and their implications to the function of light chains

Experiments have been carried out to assess the involvement of the myosin light chains [obtained by treatment of myosin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2)] in the control of cross-bridge movement and actomyosin interactions. Chymotryptic digestions of myosin, actomyosin, and myofibrils do not detect any Ca2+-induced change in the subfragment 2 region of myosin. Actin, like Ca2+, protects the in situ Nbs2 light chains from proteolysis and causes a partial switch in the digestion product of myosin from subfragment 1 to heavy meromyosin. This effect is independent of the state of aggregation of myosin, and it persists in acto heavy meromyosin and in actinomyosin in 0.6 M NaCl. Digestions and sedimentation studies indicate that there is no direct acto light chain interaction. Proteolysis of myosin shows a gradual transition from production of heavy meromyosin to subfragment 1 with lowering of the salt level. In the presence of Ca2+ heavy meromyosin is generated both in digestions of polymeric and of monomeric myosin. These results are explained in terms of localized changes within the Nbs2 light chains and subfragment 1. Subunit interactions in the myosin head lead to a Ca2+-induced reduction in the affinity of heavy meromyosin for actin in the presence of MgATP. The resulting Ca2+ inhibition of the actin-activated ATPase of myosin can be detected at high salt concentrations(75 mM KCl).     

Isoforms of tau protein from mammalian brain and avian erythrocytes: Structure, self-assembly, and elasticity

Previous studies on tau protein showed that the protein forms paracrystals which are unusually elastic. The paracrystals were obtained from a mixture of isoforms prepared from brain tissue, and the protein was in a mixed state of phosphorylation. Subsequently we showed that the structure and elasticity was related to the state of phosphorylation. However, this left open the possibility that the isotype composition played a role as well. We have now addressed this question by separating the individual isoforms and analyzing their structure. The paracrystals from all isoforms are similar to one another and to those of the native mixture; the same holds for the elasticity. Thus the tendency to self-associate, the apparent structure, and the elasticity are determined by those regions of tau which all isoforms have in common. In addition we compare tau paracrystals from three different sources. Apart from the porcine brain tau described earlier we have prepared paracrystals from bovine brain tau because its sequence is now known (Himmler et al., 1989). The structure and elasticity is indistinguishable from porcine tau. Second, we have prepared tau from avian erythrocytes where it is found in the membrane-associated marginal band microtubules ( Murphy and Wallis, 1985). Its isoform composition differs from mammalian brain tau, but again the structural properties are similar. A notable difference is that the shift in electrophoretic mobility induced by phosphorylation with CaM kinase, typical of all brain tau isotypes, is not found in the marginal band tau. Tau shows a strong tendency of longitudinal self-association which is apparent not only in the crystallization buffer but also in standard microtubule reassembly buffer. This leads to rod-like tau oligomers, fibers, and three-dimensional networks. This property, coupled with tau's elasticity, suggests a role in the organization of the cytoplasm beyond the stabilization of microtubules.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.