Melting of myosin rod as revealed by electron microscopy. II. Effects of temperature and pH on length and stability of myosin rod and its fragments

Effects of temperature and pH on intact rabbit and chicken myosin, isolated myosin rods, rabbit subfragment-2 (61 kDa, 53 kDa, and 34 kDa) and chicken light meromyosin (LMM) fragments were tested to induce a phase transition from alpha-helix to coil conformation, within the hinge region. The influence of temperature and pH were studied directly with length determination by electron microscopy. An increase of temperature to 50 degrees C yielded a shortening of 16 nm, 8 to 9 nm and 7 to 11 nm for intact myosin, isolated rods and long S-2 fragments, respectively. The length of the 34 kDa short S-2 and LMM fragments were unchanged. An increase of pH from neutral to pH 8.0 yielded values that were somewhat smaller, e.g. 12 nm, 6 nm and 6 to 8 nm for intact myosin, isolated rods and long S-2 fragments, respectively, whereas the 34 kDa short S-2 LMM fragments were also unaffected. Thus, melting and subsequent shortening is confined to the region between LMM and short S-2 segment, that is the hinge region. Alteration of temperature had a stronger shortening effect than alteration of pH, and shortening of long S-2 was more pronounced under physiological salt conditions as compared with high (0.3 M) salt. The shortening of rods in intact myosin amounted to twice the value observed with isolated rods. The amount of contraction was somewhat smaller in rods than in the 61 kDa and 53 kDa long S-2 fragments.     

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.     

The myosin filament. XIII. The sensitivity of LMM assembly to Mg.ATP

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

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).     

Transport properties of rigid bent-rod macromolecules and of semiflexible broken rods in the rigid-body treatment. Analysis of the flexibility of myosin rod.

The translational diffusion coefficients, rotational relaxation times and intrinsic viscosities of rigid bent rods, composed by two rodlike arms joined rigidly at an angle alpha, have been evaluated for varying conformation using the latest advances in hydrodynamic theory. We have considered semiflexible rods in which the joint is an elastic hinge or swivel, with a potential V(alpha) = 1/2Q alpha 2 with constant Q. Accepting the rigid-body treatment, we calculate properties of broken rods by averaging alpha-dependent values for rigid rods. The results are finally used to interpret literature values of the properties of myosin rod. Q is regarded as an adjustable parameter, and the value fitted is such that the average bending angle of myosin rod is approximately 60 degrees.     

Electric birefringence study of rabbit skeletal myosin subfragments HMM, LMM, and rod in solution.

Electric birefringence measurements and depolarized light scattering experiments were performed with HMM, LMM, and rod, the three fragments of myosin, under conditions (0.3 M KCl, 0.02 M PO4, pH 7.3) the medium currently used for biochemical assays of myosin in its native state as well as of its subfragments. The comparison of myosin and rod relaxation times (17.2 and 22.8 microseconds, respectively) suggests that the average bend angle in the tail is sharper in intact myosin (90 degrees) whereas rod, when detached from the heads, is a more elongated species with an average bend angle of 120-135 degrees. The LMM relaxation time (6.4 microseconds) is consistent with a rigid linear stick model of length 78 nm. Flexibility in myosin tail is thus confirmed as located in the HMM-LMM hinge. LMM and rod did not exhibit any significant variation of their apparent relaxation times with concentration and the decay curves were best fitted by a single exponential, evidence that the concentration of parallel staggered dimers was negligible in the concentration range studied here (0-7 g/l). This observation lends support to previous results obtained with myosin. Respective HMM, LMM, and rod molecular weights and homogeneity as evaluated by SDS-PAGE analysis were correlated to the Kerr constants of their solutions. Large variations in LMM Kerr constants could be related to the loss of a COOH-terminal peptide on prolonged chymotryptic digestion. Electric birefringence combined with depolarized light scattering is presented as a potential method for net charge distribution studies.     

Localization of a new proteolytic site accessible in oxidized myosin rod

We have compared the proteolysis pattern of reduced and oxidized myosin rods in which the five pairs of SH-groups were interchain crosslinked by employing CuCl2 or 5,5'-dithiobis-2-nitrobenzoate. In the tryptic digest of oxidized rod three new fragments appeared on SDS-polyacrylamide gel electrophoresis (chain masses of 100, 45, and 25 kDa). Based on the N-terminal sequences of the isolated peptides, it is concluded that oxidation creates a new cleavage site 102 residues away from the N-terminus of the rod, in the vicinity of one of the modified SH-groups (Cys-108). This observation indicates that S-S crosslinking of myosin rod leads to a local unfolding of the coiled-coil structure.     

Flexibility of Acanthamoeba myosin rod minifilaments.

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.     

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.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

Thermal stability of myosin rod from various species

The radius of gyration and fraction helix as a function of temperature have been determined for myosin rod from four different species: rabbit, frog, scallop, and antarctic fish. Measurements from sodium dodecyl sulfate gel electrophoresis indicate that all particles have the same molecular weight (approximately 130K). All fragments are nearly 100% alpha-helical at low temperatures (0-5 degrees C). The melting profiles for each are qualitatively similar in shape, but their midpoints are shifted along the temperature axis in the following order: antarctic fish (Tm = 33 degrees C), scallop (Tm = 39 degrees C), frog (Tm = 45 degrees C), and rabbit (Tm = 49 degrees C). Corresponding radius of gyration vs temperature profiles for each species are shifted to lower temperatures (approximately 5-8 degrees C) with respect to the optical rotation melting curves. From plots of radius of gyration vs fraction helix, we find a marked drop in the radius of gyration (from 43 to approximately 34 nm) with less than a 5% decrease in fraction helix for rabbit, frog, and antarctic fish rods, whereas the radius of gyration of scallop rod never exceeds 34 nm. Results indicate hinging of the myosin rod of each species. The thermal stabilities of the myosin rods shift in parallel with the working temperature of their respective muscles.     

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.     

Solubility-determining domain of smooth muscle myosin rod

Chymotryptic digestion of chicken gizzard light meromyosin (LMM) produced a 72 kDa core fragment, which was fully soluble at 150 mM KCl, pH 6.5–7.5. The fragment showed weak self-association at 50 mM KCl. The homology of the N-terminus amino acid sequence of this fragment with the sequence of the rabbit skeletal myosin rod suggested that the N-terminus of the core fragment originated 5 kDa from the hinge common to both smooth and skeletal myosin rod. Sedimentation experiments indicated that the domain specifying the insolubility of the intact LMM was 13 kDa long. Progressive proteolytic shortening of this region produced LMM fragments of progressively increasing solubility. Electron microscopy of segments formed from full-length LMM and from LMM core suggested that this 13 kDa domain specified the 43 nm parallel and antiparallel molecular overlaps characteristic of self-assembled intact myosin.     

Orientation dependence of displacements by a single one-headed myosin relative to the actin filament.

Displacements of single one-headed myosin molecules in a sparse myosin-rod cofilament were measured from bead displacements at various angles relative to an actin filament by dual optical trapping nanometry. The sparse myosin-rod cofilaments, 5-8 micron long, were synthesized by slowly mixing one-headed myosin prepared by papain digestion with myosin rods at molar ratios of 1:400 to 1:1500, so that one to four one-headed myosin molecules were on average scattered along the cofilament. The bead displacement was approximately 10 nm at low loads ( approximately 0.5 pN) and at angles of 5-10 degrees between the actin and myosin filaments (near physiologically correct orientation). The bead displacement decreased with an increase in the angle. The bead displacement at nearly 90 degrees was approximately 0 nm. When the angle was increased to approximately 150 degrees-170 degrees, the bead displacements increased to 5 nm. A native two-headed myosin showed similar size and orientation dependence of bead displacements as a one-headed myosin.     

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.     

Packing of myosin molecules in muscle thick filaments

The backbone of the myosin filament is an aggregate of alpha-helical coiled coil myosin rods. Its surface forms a three-stranded helix composed of myosin heads. Currently there is no adequate model to describe the organization of the myosin filament. It is proposed here that, in cross-section the light meromyosin (LMM) of 18 myosin molecules form an outer tube, with nine S2 forming the interior core. At the surface of the thick filament, myosin heads are arranged in three rows, giving the filament a periodicity of 14.3 nm per three myosin molecules. Two of these molecules are organized at an angle of 120 degrees to each other on the same level, while the third is shifted 7.2 nm along the filament axis. This packing gives a striation pattern of 7.2 nm by electron microscopy. An alternative model is also possible, in which the heads of the myosin molecules are uniformly spaced at an interval of 14.3 nm along the filament axis. The packing of individual molecules within the myosin filament is based on a regular pattern of charge on the 28 amino-acid repeat in the rod domain.     

X-ray scattering by single-headed heavy meromyosin. Cleavage of the myosin head from the rod does not change its shape

Low angle X-ray scattering from heavy meromyosin (HMM) and from single-headed heavy meromyosin (sHMM) have been examined to determine if the heads of myosin change shape when cleaved from the rod to form subfragment 1 (S1). The scattering intensities of intact HMM and sHMM were compared with those of their chymotryptic digestion products, S1 and subfragment 2 (S2). As the data with HMM were complicated by scattering between the two heads, the more extensive analysis was done with sHMM. Pseudo-Guinier plots of intact and digested sHMM, over the angular range used previously for S1, were linear and showed a difference in apparent radius of gyration (Rg) of only 0.07 +/- 0.04 nm. The absolute apparent Rg value of sHMM was 3.2 +/- 0.2 nm, which is comparable to the radius of gyration reported previously for S1 alone. A plot of the fractional differences in scattering intensities of intact and digested sHMM was flat to a reciprocal spacing of at least 1/3.5 nm-1. These results indicate that the head portions of sHMM and S1 have very similar structures at low resolution. Scattering curves for various models of sHMM and mixtures of S1 and S2 were calculated and the fractional difference plots of scattering intensities were made to determine how sensitive this type of analysis is to changes in the shape of the head. Changes in Rg of 0.1 nm or greater gave detectably non-flat difference plots. Thus, the X-ray scattering of sHMM (and HMM) demonstrated that differences in structure between the head of myosin and isolated S1 are likely to be small. Current controversies over myosin head structure are discussed in light of this result.     

Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle.

1. The molecular weights of a series of synthetic myosin filaments have been measured, using the transport-concentration dependence theory of Rowe, A.J. [Biopolymers, 1977, 16, 2595--2611]. It is shown that for preparations of narrow length distribution (0.60--0.77 micrometer), N, the number of myosin molecules/14.3 nm varies between 3 and 6. 2. The reduced specific viscosity of synthetic myosin filaments has been measured as a function of both concentration and shear rate. From the concentration dependence at zero rate of shear, a value for the "swelling" of the filaments Vs/-v = 2.3 has been calculated. 3. The frictional coefficient of synthetic myosin filaments has been shown to be anomalously but reproducibly high, as compared to that of prolate ellipsoids of the same length and mass. This additional frictional drag has been numerically characterised by a "frictional increment", fi = 1.76 +/- 0.11. 4. A procedure has been devised whereby for any elongated structure which can be assumed to show the same (or other known) fi value, the molecular weight can be estimated from s0 (extrapolated sedimentation coefficient) and 2b (length) alone. 5. An s0 value for natural A-filaments, isolated from rabbit psoas muscle, has been determined by the active enzyme centrifugation technique. From this value, s0 = 132 +/- 3 S, a molecular weight of 1.20 . 10(8) has been computed by the new procedure, for preparations of average length 1.27 micrometer. 6. Contingent upon the validity of the assumptions used (see 4 above) the N value is computed as 3.1 +/- 0.2, consistent with the native, fully intact A-filament having three-fold symmetry, containing 294 myosin molecules, and having a molecular weight based upon myosin and C-protein of 1.31 . 10(8).