Preparation and properties of smooth muscle myosin from horse esophagus

Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle.

The ratio of the absorption of myosin S at 280 nm to that at 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/10⁵ g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KCl and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A.

Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per 10⁵ g of protein as horse and rabbit skeletal myosin A (about 8 moles/10⁵ g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline.

Ca²⁺-ATPase of myosin S in the presence of 0.5 M KCl and Mg²⁺-ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca²⁺-ATPase in the presence of 0.05 M KCl were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25°, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N-ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca²⁺-ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca²⁺-ATPase during the course of the reaction of sulfhydryl groups of myosin with N-ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A.     

The structural basis of muscle contraction

The myosin cross-bridge exists in two conformations, which differ in the orientation of a long lever arm. Since the lever arm undergoes a 60 degree rotation between the two conformations, which would lead to a displacement of the myosin filament of about 11 nm, the transition between these two states has been associated with the elementary 'power stroke' of muscle. Moreover, this rotation is coupled with changes in the active site (CLOSED to OPEN), which probably enable phosphate release. The transition CLOSED to OPEN appears to be brought about by actin binding. However, kinetics shows that the binding of myosin to actin is a two-step process which affects both ATP and ADP affinity and vice versa. The structural basis of these effects is only partially explained by the presently known conformers of myosin. Therefore, additional states of the myosin cross-bridge should exist. Indeed, cryoelectron microscopy has revealed other angles of the lever arm induced by ADP binding to a smooth muscle actin-myosin complex.     

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody

The position of the N terminus of myosin light chain 1 (LC1) and myosin light chain 2 (LC2) of rabbit skeletal muscle was mapped on the myosin head with a monoclonal antibody (SI304), which recognized the amino acid sequence N-trimethylalanyl-prolyl-lysyl-lysyl at the N terminus of LC1 and LC2. The complex of the antibody and myosin was observed by electron microscopy. By selective cleavage of the N terminus of LC1 or LC2 with papain or chymotrypsin, the position of the N terminus of LC1 and LC2 was determined separately. The N terminus of LC2 is located at the head-rod junction. The N terminus of LC1 is 11 nm (+/- 3 nm, standard deviation) from the head-rod junction. This position is near the actin-binding site of the myosin head.     

Structure of the cross-striated adductor muscle of the scallop

The structure of the cross-striated adductor muscle of the scallop has been studied by electron microscopy and X-ray diffraction using living relaxed, glycerol-extracted (rigor), fixed and dried muscles. The thick filaments are arranged in a hexagonal lattice whose size varies with sarcomere length so as to maintain a constant lattice volume. In the overlap region there are approximately 12 thin filaments about each thick filament and these are arranged in a partially disordered lattice similar to that found in other invertebrate muscles, giving a thin-to-thick filament ratio in this region of 6:1.The thin filaments, which contain actin and tropomyosin, are about 1 μm long and the actin subunits are arranged on a helix of pitch 2 × 38.5 nm. The thick filaments, which contain myosin and paramyosin, are about 1.76 μm long and have a backbone diameter of about 21 nm. We propose that these filaments have a core of paramyosin about 6 nm in diameter, around which the myosin molecules pack. In living relaxed muscle, the projecting myosin heads are symmetrically arranged. The data are consistent with a six-stranded helix, each strand having a pitch of 290 nm. The projections along the strands each correspond to the heads of one or two myosin molecules and occur at alternating intervals of 13 and 16 nm. In rigor muscle these projections move away from the backbone and attach to the thin filaments.In both living and dried muscle, alternate planes of thick filaments are staggered longitudinally relative to each other by about 7.2 nm. This gives rise to a body-centred orthorhombic lattice with a unit cell twice the volume of the basic filament lattice.     

Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone

The site of photocross-linking between Cys-697 (SH2), prelabeled with 4'-[14C]maleimidylbenzophenone, and the 50-kDa segment of myosin S1 on irradiation in the absence of nucleotide has been determined by isolation of the 20-50-kDa adduct and subsequent tryptic proteolysis. Isolation and partial sequencing of the radioactively labeled peptide indicate that the site of cross-linking is Arg-239. This result indicates that, in the absence of nucleotide, Arg-239 resides at about 1.0 nm from SH2 and, on the basis of the recent work of Sutoh and Hiratsuka (Sutoh, K. and Hiratsuka T. (1988) Biochemistry 27, 2964-2969) places Arg-239 at no more than 1.45 nm from either Lys-184 or Lys-189 of the nucleotide-binding "glycine-rich" loop prior to the binding of nucleotide.     

LC2 involvement in the assembly of skeletal myosin filaments

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.     

Studies of the interaction between titin and myosin

The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH- terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.     

Direct observation of the myosin Va recovery stroke that contributes to unidirectional stepping along actin

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed "strokes"; the "power stroke" is the force-generating swinging of the myosin light chain-binding "neck" domain relative to the motor domain "head" while bound to actin; the "recovery stroke" is the necessary initial motion that primes, or "cocks," myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a "hand over hand" mechanism in which the trailing head detaches and steps forward ～72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ～40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥ 5 k(B)T of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va.     

19F NMR study of the myosin and tropomyosin binding sites on actin

Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz 19F NMR spectrum were unaffected by actin polymerization. However, the binding of the relaxing protein tropomyosin resulted in the fluorinated Lys-61 resonances broadening out beyond detection due to a substantial increase in the effective correlation time of the label. Similarly, the binding of myosin subfragment 1 to F-actin resulted in the dramatic broadening of the labeled Lys-61 resonances. Thus, Lys-61 on actin appears to be closely associated with the binding sites for both tropomyosin and myosin, suggesting that both these proteins can compete for the same site on actin. The other region of actin known to be involved in myosin binding, Cys-10, was found to be more remote from the actin-actin interfaces than Lys-61. Labels on Cys-10 exhibited substantially greater mobility than fluorescein 5-isothiocyanate attached to Lys-61 which appeared to be held down on the surface of the actin monomer. This may sterically hinder the actin-actin interaction about 1 nm from the tropomyosin/myosin binding site.     

MgATP binding changes neither the shape nor the flexibility of the heads of single myosin molecules.

The structural mechanism by which myosin heads exert force is unknown. One possibility is that the tight binding of the heads to actin drives them into a force-generating configuration. Another possibility is that the force-generating conformational change is inherent to the myosin heads. In this case the heads would make force by changing their shape according to the species of nucleotide in their active sites, the tight attachment to actin serving only to provide traction. To test this latter possibility, we used negative stain electron microscopy to search for a MgATP-induced shape change in the heads of single myosin molecules. We compared the heads of 10S smooth muscle myosin monomers (wherein MgATP is trapped at the active site) with the MgATP-free heads of 6S monomers. We found that to a resolution of about 2 nm, MgATP binding to the unrestrained myosin head does not drive it to change its shape or its flexibility. This result suggests that the head makes force by virtue of an induced fit to actin.     

Plasma membrane association of Acanthamoeba myosin I

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F- actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI- extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP- sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin- binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.     

Studies on the physical states of human platelet myosin in crude extracts

The physical properties of human platelet myosin in crude extracts were studied by means of Sepharose 4B gel filtration and sucrose density gradient centrifugation in the presence or absence of Mg-ATP. Platelet myosin extracted with a buffer containing 0-0.15 M KCl gave a Stokes radius of about 12.0-12.5 nm irrespective of the presence or absence of Mg-ATP. The sedimentation coefficients obtained in the presence of Mg-ATP were about 10-11 and 8.5S at 0.05-0.10 and 0.15 M KCl, respectively, whereas the values obtained in the absence of Mg-ATP were about 16, 9-12, and 8.5S at 0.05, 0.10, and 0.15 M KCl, respectively. The apparent molecular weight in the presence of Mg-ATP, therefore, was about 500,000 and 420,000 at 0.05-0.10 and 0.15 M KCl, respectively, while the molecular weight in the absence of Mg-ATP was about 790,000, 460,000-620,000, and 440,000 at 0.05, 0.10, and 0.15 M KCl, respectively. The purified monomeric platelet myosin that had been solubilized with Mg-ATP at 0.10 M KCl had a Stokes radius of about 12.5 nm, a sedimentation coefficient of about 9S, and an apparent molecular weight of 460,000. On the other hand, while crude platelet myosin extracted at 0.6 M KCl with Mg-ATP gave a Stokes radius of about 20 nm, a sedimentation coefficient of about of 6S, and an apparent molecular weight of about 490,000, each of these physical parameters obtained in the absence of Mg-ATP was much larger than that obtained in the presence of Mg-ATP because the myosin was associated with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 microscopic visualization of the N terminus of the myosin heavy chain using a site-directed antibody

We determined the spatial location of the N terminus of the heavy chain of rabbit skeletal muscle myosin by electron microscopy, using a site-directed antibody raised against its N-terminal eight residues as an electron microscopic probe. By examining rotary-shadowed images of the heavy meromyosin-antibody complex, we measured distances between the head-rod junction and the attachment site of the antibody bound on the head. The average distance was estimated to be about 12 nm. The result indicates that the N terminus of the heavy chain is located at the middle region of the head.     

Hinging of rabbit myosin rod

The question of hinging in myosin rod from rabbit skeletal muscle has been reexamined. Elastic light scattering and optical rotation have been used to measure the radius of gyration and fraction helix, respectively, as a function of temperature for myosin rod, light meromyosin (LMM), and long subfragment 2 (long S-2). The radius of gyration vs temperature profile of myosin rod is shifted with respect to the optical rotation melting curve by about -5 degrees C. Similar studies on both LMM and long S-2 show virtually superimposable profiles. To correlate changes in the secondary structure with the overall conformation, plots of radius of gyration vs fraction helix are presented for each myosin subfragment. Myosin rod exhibits a marked decrease in the radius of gyration from 43 nm to approximately 35 nm, while the fraction helix remains at nearly 100%. LMM and long S-2 did not show this behavior. Rather, a decrease in the radius of gyration of these fragments occurred with comparable changes in fraction helix. These results are interpreted in terms of hinging of the myosin rod within the LMM/S-2 junction.     

The myosin filament. VI. Myosin content.

There has been some disagreement about the number of myosin molecules in vertebrate skeletal myosin filaments calculated from the myosin to actin weight ratio determined by quantitative sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Tregear &; Squire, 1973; Potter, 1974; Morimoto &; Harrington, 1974). In this work it was found that (1) thoroughly washed fibrils are required to obtain the true value for the myosin to actin weight ratio. (2) Neither actin nor myosin is extracted preferentially during the required washing procedure. (3) There are four myosin molecules per 14.3 nm interval along the myosin filament or about 400 myosin molecules per filament.From published estimates of the number of molecules of C-protein per myosin filament (Offer et al., 1973; Morimoto &; Harrington, 1974) and the findings in this work, we conclude that there are four molecules of C-protein at each of the 14 C-protein binding positions along the filament, i.e. one C-protein molecule for each of the four myosin molecules contributing to the cross-bridges at each position.     

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.