Cryo-atomic force microscopy of smooth muscle myosin.

The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

Electron microscopy of negatively stained scallop myosin molecules. Effect of regulatory light chain removal on head structure

The heads of myosin molecules from the striated adductor muscle of scallop have been studied by electron microscopy after negative staining. In common with vertebrate skeletal muscle myosin visualized by this method, the scallop myosin heads were pear-shaped and often showed pronounced curvature. Staining suggestive of two or, more frequently, three domains could often be observed. Removal of regulatory light chains (R-LCs) resulted in a reduction in the length of the heads of about 2.6 nm, with no significant change in maximum width. In desensitized preparations a majority of heads displayed anticlockwise curvature, whereas intact heads were usually seen curved clockwise. Analysis of the head curvature in both intact and desensitized molecules was consistent with an ability of each head to rotate about its long axis. Desensitization resulted in an increased incidence of heads showing two domains. It seems likely that the reduction in length upon removal of the R-LC is due to the two small domains located in the neck region of the head collapsing into one.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.     

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.     

Morphological aspects of thiophosphorylated and dephosphorylated myosin molecules from the plasmodium of Physarum polycephalum

Electron microscopically, the myosin molecule from the plasmodium of Physarum polycephalum has a long tail of 173 nm, having a flexible region over the range of 80 to 120 nm from the head-tail junction. In 0.6 M ammonium acetate, this region of the dephosphorylated myosin molecules is more flexible than that of the thiophosphorylated ones. In 50 mM ammonium acetate, the dephosphorylated myosin molecules exist in monomeric and oligomeric forms, independently of ATP and Mg2+, whereas the thiophosphorylated myosin molecules form dense aggregates of thick filaments. The tails of the monomeric dephosphorylated myosin molecules bend sharply at the flexible region at angles of more than 120 degrees. In oligomers of the dephosphorylated myosin molecules, the molecules are all associated side-to-side with straight tails and are oriented in the same direction. Based on these results, the regulation mechanism of cell motility of the plasmodium is discussed.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Electric birefringence study of the solution structure of chymotrypsin-cleaved Acanthamoeba myosin II

After removal of the 66 COOH-terminal amino acids from each of its two heavy chains by chymotrypsin digestion, Acanthamoeba myosin II forms only parallel dimers under conditions in which native myosin II forms bipolar filaments (Kuznicki, J., Cote, G. P., Bowers, B., and Korn, E. D. (1985) J. Biol. Chem. 260, 1967-1972). We have studied the solution structure of the chymotrypsin-cleaved myosin II by electric birefringence. Only two species, known to be monomer and parallel dimer from previous studies, were detected. The contribution to the birefringence decay from dimer increased from about 10 to 70% as the KCl concentration was lowered from 100 mM to 0 in 50% glycerol. At all ionic strengths, the monomer had a relaxation time corrected to water at 20 degrees C of 8.2 microseconds, whereas a relaxation time of 10.3 microseconds was expected for monomers with straight rigid rods. This strongly indicates that the myosin rod in solution is bent. On the assumption that there is a single bend 26 nm from the tip of the tail, as suggested by electron microscopy, it was calculated that the average bend angle would be 110 degrees, in solution, if as seems most likely, the average angle between the two globular heads were 180 degrees. The observed relaxation time of the dimer corrected to water at 20 degrees C was 25 microseconds, independent of ionic strength, which, if the motion of the heads were unrestricted, is consistent with a structure for a parallel dimer in which either the two monomer subunits have straight rigid rods and are staggered by about 28 nm or only one is bent and the stagger is 30 nm. As described in the accompanying Appendix, either of these dimers can be assembled into a bipolar filament compatible with the apparent structure of filaments of native myosin II (Pollard, T.D. (1982) J. Cell Biol. 95, 816-825).     

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).     

Bending of smooth muscle myosin rod

Electron microscopy of mammalian smooth muscle myosin rods showed them to be 153 +/- 7 nm (SD) long, and to bend sharply (greater than 90 degrees) but infrequently, and pH independently (range 6.5-9.5), at a single site 45 +/- 4 nm from one end of the molecule. Light meromyosin (LMM) preparations were 99 +/- 10 nm long, and showed no bends. Intrinsic viscosity vs temperature plots for rods and LMM indicated that neither fragment changed in flexibility in the range 4-40 degrees C. Peptide mapping in the presence and absence of SDS established that the proteolytic susceptibility of the hinge at the N terminus of LMM reflects the presence of locally different structure, and not simply a clustering of susceptible residues. The isolated smooth muscle myosin rod thus contains only a single hinge, having significant stiffness, and lacks the second bend seen under certain conditions in the intact molecule.     

Electron microscope study of the effect of temperature on the length of the tail of the myosin molecule

The effect of temperature on the length of the tail of the myosin molecule has been studied by negative staining of molecules immobilized on carbon substrates at different temperatures. In buffers containing chloride as the principal anion, tail length was approximately constant up to 25 degrees C. Above this temperature, it shortened linearly with increasing temperature up to 42 degrees C, the highest temperature studied in this solvent. The amount of shortening per degree C was about 1.2 nm. A similar amount of shortening per degree C was seen in acetate-containing buffers up to 50 degrees C, but in this case it did not begin until the temperature exceeded about 40 degrees C. A large fraction of the observed shortening was localized in a region that lies roughly between the two positions in the tail where proteolysis results in production of short or long subfragment-2. Frequently, the tail had a different appearance in this region from elsewhere and could sometimes be seen to split into two strands that were separate but coiled around one another.     

A new model for the surface arrangement of myosin molecules in tarantula thick filaments

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head.We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing.Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

Modulation of intramolecular interactions in superhelical DNA by curved sequences: a Monte Carlo simulation study.

A Monte Carlo model for the generation of superhelical DNA structures at thermodynamic equilibrium (Klenin et al., 1991; Vologodskii et al., 1992) was modified to account for the presence of local curvature. Equilibrium ensembles of a 2700-bp DNA chain at linking number difference delta Lk = -15 were generated, with one or two permanent bends up to 120 degrees inserted at different positions. The computed structures were then analyzed with respect to the number and positions of the end loops of the interwound superhelix, and the intramolecular interaction probability of different segments of the DNA. We find that the superhelix structure is strongly organized by permanent bends. A DNA segment with a 30 degrees bend already has a significantly higher probability of being at the apex of a superhelix than the control, and for a 120 degrees bend the majority of DNAs have one end loop at the position of the bend. The entropy change due to the localization of a 120 permanent bend in the end loop is estimated to be -17 kJ mol-1 K-1. When two bends are inserted, the conformation of the superhelix is found to be strongly dependent on their relative positions: the straight interwound form dominates when the two bends are separated by 50% of the total DNA length, whereas the majority of the superhelices are in a branched conformation when the bends are separated by 33%. DNA segments in the vicinity of the permanent bend are strongly oriented with respect to each other.     

Shape and flexibility of the myosin molecule

Molecules of rabbit skeletal myosin have been examined in the electron microscope after drying at low temperature from solutions containing ethylene glycol or glycerol and rotary-shadowing with platinum. Analysis of the structure has been assisted by stereo-photography. While the general appearance, two heads attached to a long tail, is similar to that described by Slayter & Lowey (1967), more detail about the shape and size of the heads can be discerned and new information has been obtained about the flexibility of the tail and the head-tail junction.The heads are 190 Å long and wider at their ends than near the junction with the tail; the shape resembles that of a pear. The length is appreciably greater than the generally accepted value for subfragment 1, the proteolytic fragment of myosin. The heads are flexibly attached to the tail and can assume a wide range of tilt angles.Because the point where the two heads join the tail can be identified, the length of the tail, 1560 (±50) Å, can be measured more accurately than formerly. While all parts of the tail are somewhat flexible, sharp bends often occur at a well-defined site 430 Å from the head-tail junction. The demonstration of hinges at the head-tail junction and in the tail provides strong support for H. E. Huxley's (1969) hypothesis for the mechanism of muscle contraction.     

Identification and characterization of Tetrahymena myosin

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K⁺-/NH₄⁺-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain.     

Myosin head configuration in relaxed insect flight muscle: x-ray modeled resting cross-bridges in a pre-powerstroke state are poised for actin binding

Low-angle x-ray diffraction patterns from relaxed insect flight muscle recorded on the BioCAT beamline at the Argonne APS have been modeled to 6.5 nm resolution (R-factor 9.7%, 65 reflections) using the known myosin head atomic coordinates, a hinge between the motor (catalytic) domain and the light chain-binding (neck) region (lever arm), together with a simulated annealing procedure. The best head conformation angles around the hinge gave a head shape that was close to that typical of relaxed M*ADP*Pi heads, a head shape never before demonstrated in intact muscle. The best packing constrained the eight heads per crown within a compact crown shelf projecting at approximately 90 degrees to the filament axis. The two heads of each myosin molecule assume nonequivalent positions, one head projecting outward while the other curves round the thick filament surface to nose against the proximal neck of the projecting head of the neighboring molecule. The projecting heads immediately suggest a possible cross-bridge cycle. The relaxed projecting head, oriented almost as needed for actin attachment, will attach, then release Pi followed by ADP, as the lever arm with a purely axial change in tilt drives approximately 10 nm of actin filament sliding on the way to the nucleotide-free limit of its working stroke. The overall arrangement appears well designed to support precision cycling for the myogenic oscillatory mode of contraction with its enhanced stretch-activation response used in flight by insects equipped with asynchronous fibrillar flight muscles.     

Direct observation of molecular motility by light microscopy

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.