Substructure and accessory proteins in scallop myosin filaments

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.     

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.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.     

Millisecond time-resolved changes occurring in Ca2+-regulated myosin filaments upon relaxation

Contraction of many muscles is activated in part by the binding of Ca²⁺ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca²⁺ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca²⁺ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca²⁺ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process.     

Electron microscopy and image analysis of myosin filaments from scallop striated muscle

Thick filaments have been isolated from the striated adductor muscle of the scallop and examined by electron microscopy after negative staining. Many filaments appear intact, and reveal a centrally located bare-zone and a well-defined helical surface array of myosin crossbridges characterized by a 145 A axial period and prominent helical tracks of pitch 480 A. Heavy-metal shadowing shows that these helices are right-handed. A small perturbation of alternate crossbridge levels produces an axial period of 290 A, which is most prominent in a region on either side of the bare-zone. Image analysis reveals that the crossbridge array has 7-fold rotational symmetry, one of the possibilities suggested by earlier X-ray diffraction studies of native filaments in scallop muscle. A low-resolution three-dimensional reconstruction shows elongated surface projections ("crossbridges") that probably represent unresolved pairs of myosin heads. They run almost parallel to the filament surface, but are slewed slightly from the axis so that they lie along the right-handed helical tracks of pitch 480 A. The connection to the filament backbone probably occurs at the end of the crossbridges nearer the bare-zone; thus, their sense of tilt appears to be opposite to that of rigor attachment to actin. The 290 A period arises from a different distribution of crossbridge density at alternate levels; in addition, there are weak connections between the top of one crossbridge and the bottom of the next, 145 A away. The prominence of the 290 A period near the bare-zone suggests that anti-parallel molecular interactions are mainly responsible for this perturbation.     

Fluorescence measurements detect changes in scallop myosin regulatory domain.

Ca2+-induced conformational changes of scallop myosin regulatory domain (RD) were studied using intrinsic fluorescence. Both the intensity and anisotropy of tryptophan fluorescence decreased significantly upon removal of Ca2+. By making a mutant RD we found that the Ca2+-induced fluorescence change is due mainly to Trp21 of the essential light chain which is located at the unusual Ca2+-binding EF-hand motif of the first domain. This result suggests that Trp21 is in a less hydrophobic and more flexible environment in the Ca2+-free state, supporting a model for regulation based on the 2 A resolution structure of scallop RD with bound Ca2+ [Houdusse A. and Cohen C. (1996) Structure 4, 21-32]. Binding of the fluorescent probe, 8-anilinonaphthalene-1-sulphonate (ANS) to the RD senses the dissociation of the regulatory light chain (RLC) in the presence of EDTA, by energy transfer from a tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We identified a hydrophobic pentapeptide (Leu836-Ala840) at the head-rod junction which is required for the effective energy transfer and conceivably is part of the ANS-binding site. Extension of the HC component of RD towards the rod region results in a larger ANS response, presumably indicating changes in HC-RLC interactions, which might be crucial for the regulatory function of scallop myosin.     

Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.     

Ca2+ activates actin-filament sliding on scallop myosin but inhibits that on Physarum myosin

The actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by microM levels of Ca2+, the mode of which is in contrast to the activating effect of Ca2+ on scallop myosin (Kohama, K. (1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.     

Structural studies of synthetic filaments prepared from column-purified myosin.

Synthetic filaments prepared from column-purified rabbit skeletal myosin by slow dialysis exhibit characteristic bipolar organization and 14-nm axial subunit spacing. Backbone substructure can be discerned in high resolution micrographs in the form of striations of 3--4-nm width and slight angular tilt from the direction of the filament axis. Filament backbone diameters vary over the population, although remaining relatively constant for a single filament. Approximately 25% of the filaments appear poorly stained and frayed, which may be due to collapse on the electron microscope grid. Optical diffraction studies reveal a 43-nm axial repeat as well as the 14.3-nm subunit repeat, indicating a structural homology with natural filaments. A model for synthetic filament aggregation is presented that is consistent with observations of backbone diameter variation, absence of bare zones, and the presence of fraying filaments.     

Structural changes in trichocyte keratin intermediate filaments during keratinization

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.     

Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.     

Vectorial activation of smooth muscle myosin filaments and its modulation by telokin

Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.     

Essential light chain exchange in scallop myosin

The exchange of essential light chains (SH-LCs) of scallop myosin was followed with the aid of scallop SH-LC alkylated with 14C-labeled iodoacetate. More than 70% of the SH-LCs were exchanged in myosin preparations that were desensitized by removal of both regulatory light chains (R-LCs) with ethylenediaminetetraacetic acid (EDTA) treatment. Although desensitized myosin solubilized with 0.6 M NaCl or with 10 mM adenosine 5'-triphosphate (ATP) in the absence of salt equilibrated rapidly with SH-LCs even in the cold, exchange in myosin filaments required elevated temperatures. Equilibration of the SH-LCs in desensitized preparations did not depend on ATP or magnesium ions but was significantly accelerated by actin. The desensitized myosin preparations containing alkylated SH-LCs (approximately 1 mol of thiol alkylated/mol of SH-LC) readily recombined with R-LCs. The preparations regained fully the calcium dependence of the actin-activated magnesium adenosinetriphosphatase (Mg-ATPase), contained R-LCs and SH-LCs in equimolar amounts, and had an ATPase activity similar to that of untreated myosin preparations. R-LCs interfered with the equilibration of the SH-LCs. In intact myosin preparations, the exchange of SH-LCs was slow and was frequently associated with the dissociation of the R-LCs. The blocking action of the R-LC on SH-LC exchange agrees with evidence showing that the two light chain types interact and suggests that parts of the SH-LC may lie between the R-LC and the heavy chain of myosin.     

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.     

Behavior of caldesmon upon interaction of thin filaments with myosin subfragment 1 in ghost fibers

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7 degrees and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-V(i). The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Irreversible changes occur in chromatin structure upon dissociation of histone H1

The role of histone H1 in the actual interactions bringing about chromatin folding is investigated by studying the reversibility of its dissociation. H1 was dissociated by increase of the NaCl concentration and reassociated by dialysis, without removal from the dialysis bag. To scrutinize the fidelity of this stoichiometric form of chromatin reconstitution, we use circular dichroism, nuclease digestion, thermal denaturation and the sensitive electric birefringence method. No alteration of the repeat length and no nucleosomal sliding are observed upon the reassociation procedure. However, under all the different conditions investigated, the original value of the positive electric birefringence is never recovered, indicating an irreversible change of structure. CD and melting profiles confirm that DNA-protein interactions are modified, and orientational relaxation time measurements indicate that these structural perturbations affect the salt-induced transition of polynucleosomal fibers. The striking conclusion of these studies is that variations of ionic concentration are sufficient to induce irreversible structural alterations affecting the higher-order folding of chromatin. It is of interest that the only sample which exhibits behavior upon reassociation comparable to that of native chromatin is the one which experienced the fastest salt transitions. We suggest that these conformational changes arise from the unbinding to DNA of certain basic tails of histone(s), and that a competition for DNA binding locations exists upon the reassociation. These results are then additional arguments (Mazen, A., Hacques, M.F. and Marion, C.,J. Mol. Biol. 194, 741-745 (1987)), to suggest that dissociation of H1 might modify a direct interaction between basic tails of core histones and H1.     

Conformational changes that occur during an RNA-editing adenosine deamination reaction

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.