New method to prepare single-headed heavy meromyosin with high purity and a high yield

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.     

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 binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

Structure of the myosin head in solution and the effect of light chain 2 removal.

Structural properties of rabbit skeletal myosin head (S1) and the influence of the DTNB light chain (LC2) on the size and shape of myosin heads in solution were investigated by small angle x-ray scattering. The LC2 deficient myosin head, S1 (-LC2), and the S1 containing LC2 light chain, S1 (+LC2) were studied in parallel. The respective values of the radius of gyration were found to be (40.2 +/- 0.5) A and (46.7 +/- 1) A, while the maximum dimension was (190 +/- 15) A for both species. The large difference between the two Rg values suggest that LC2 is located close to one extremity of the myosin head, in agreement with most electron microscopy observations. All models derived from the x-ray scattering pattern of the native myosin head share a common overall morphology, showing two main regions, an asymmetric globular portion which tapers smoothly into a thinner domain of roughly equivalent length making an angle of approximately 60 degrees, with a contour length of approximately 210 A.     

Solution structure of heavy meromyosin by small-angle scattering

Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.     

Structure of myosin subfragment 1 from low-angle X-ray scattering

The X-ray scattering pattern produced by a solution of myosin subfragment 1 has been measured to a resolution (Bragg spacing) of 2 nm. We find that for subfragment 1 (S1) prepared by limited papain digestion in the presence of ethylenediaminetetraacetate the radius of gyration is 3.28 +/- 0.06 nm, the volume is 151 +/- 6 nm3, the surface area is 330 +/- 15 nm2, and the length of the maximum chord is 12.0 +/- 1.0 nm. The theoretical scattering patterns from several objects of uniform electron density have been calculated and compared with the observed scattering produced by S1. The recent three-dimensional electron micrograph reconstruction of S1-decorated actin by J. Seymour and E. O'Brien (private communication) generated the calculated pattern that best fit the observed scattering. This fit strongly suggests that this reconstruction resembles subfragment 1. The good correspondence between an S1 structure derived when S1 is attached to actin and a study of free S1 in solution strongly suggests that binding to actin does not grossly distort the shape of S1. This is consistent with the notion that S1 changes its orientation on actin, rather than its shape, in order to generate the contractile force in muscle.     

A mutant heterodimeric myosin with one inactive head generates maximal displacement

Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.     

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.     

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of affinity modification of myosin ATPase under the action of monoaldehyde derivatives of ADP]

It was shown that the highly purified monoaldehyde derivative of ADP obtained by partial reduction of the dialdehyde derivative of ADP causes strong irreversible inhibition of the Ca-ATPase activity of myosin subfragment I, the inhibiting effect being of the affinity modification type. The addition to the reaction medium of Mg2+ (but not Ca2+) during the subfragment I interaction with the inhibitor fully prevents the inhibiting effect at all substrates used (Ca-, Mg- or K, EDTA-ATPases). Contrariwise, the subfragment I modified in the absence of Mg2+ exhibits the same degree of inhibition for all the three types of the ATPase activity. An unexpected result that was previously unobserved for other affinity modifiers of myosin ATPase is the maintenance of activity in 50% of active centers, when "two-head" forms of the enzyme (the myosin proper and heavy meromyosin, HMM) are modified. Noteworthy that the affinity modification reaction is characterized by the same values of inhibition constants as in the case of myosin subfragment I (Ki = 3.3-3.5 X 10(-4) M; ki = 0.03-0.04 min-1). This finding provides additional evidence in favour of functional asymmetry of myosin heads in the myosin molecule which seems to be due to the screening of the active center of one head by the other one.     

Phosphorylation of a single head of smooth muscle myosin activates the whole molecule

Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin II, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single-turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation of one RLC abolishes the asymmetric inhibited state formed by dephosphorylated myosin [Liu, J., et al. (2003) J. Mol. Biol. 329, 963-972], allowing activation of both the phosphorylated and dephosphorylated heads. These findings help explain how smooth muscles are able to generate high levels of stress with low phosphorylation levels.     

Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin

The thermal unfolding of rabbit skeletal heavy meromyosin (HMM), myosin subfragment 1, and subfragment 2 has been studied by differential scanning calorimetry (DSC). Two distinct endotherms are observed in the DSC scan of heavy meromyosin. The first endotherm, with a Tm of 41 degrees C at pH 7.9 in 0.1 M KCl, is assigned to the unfolding of the subfragment 2 domain of HMM based on scans of isolated subfragment 2. The unfolding of the subfragment 2 domain is reversible both in the isolated form and in HMM. The unfolding of subfragment 2 in HMM can be fit as a single two-state transition with a delta Hvh and delta Hcal of 161 kcal/mol, indicating that subfragment 2 exists as a single domain in HMM. The unfolding of subfragment 2 is characterized by an extraordinarily large delta Cp of approximately 30,000 cal/(deg.mol). In the presence of nucleotides, the high-temperature HMM endotherm with a Tm of 48 degrees C shifts to higher temperature, indicating that this peak corresponds to the unfolding of the subfragment 1 domain. This assignment has been confirmed by comparison with isolated subfragment 1. The stabilizing effect of AMPPNP was significantly greater than that of ADP. The vanadate-trapped ADP species was slightly more stable than M.AMPPNP with a Tm at 58 degrees C. The unfolding of subfragment 1, both in the isolated form and in HMM, was irreversible. Only a single endotherm was noted in the DSC scans of the subfragment 1 domain of HMM and in freshly prepared subfragment 1 complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Heavy meromyosin binds actin with negative cooperativity.

The association of fluorescently labeled heavy meromyosin (HMM) and F-actin was measured by time-resolved fluorescence depolarization. The effects of varying the protein concentrations, temperature, KCl concentration, and pH were determined. Measurements of HMM mobility supported a model of no interaction between the two heads in the absence of actin. Measurements of actin binding, when compared with results for myosin subfragment I, indicated that the two heads of HMM do not bind independently in the rigor complex. This could result from actin-transmitted negative cooperativity or from steric inhibition due to the structure of HMM. For HMM and actin in 0.15 7 kcl at 25 degrees C: Ka = 3.9 X 10(7) M-1, deltaHco' = 36 +/- 2 J M-1, deltaSco' = 0.26 +/- 0.02 kJ M-1 K-1; the slope of ln Ka vs. [KCl]1/2 = -3.88 and the pH of maximum association was 6.9.     

Two functional heads are required for full activation of smooth muscle myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.     

A synthetic peptide of the N-terminus of actin interacts with myosin

Research reported from numerous laboratories suggested that the N-terminal region of actin contained one of the binding sites between actin and myosin. A synthetic peptide corresponding to residues 1-28 of skeletal actin was prepared by solid-phase peptide methodology. The formation of a complex between this peptide and myosin subfragment 1 (S1) was demonstrated by high-performance size-exclusion chromatography (pH 6.8). The actin peptide precipitated S1 at higher pH (7.4-8.2) but remained soluble when bound to heavy meromyosin (HMM) or S1 in the presence of F-actin. The actin peptide 1-28 bound to S1 and HMM and activated the ATPase activity in a manner similar to that of F-actin. These results demonstrate that the N-terminal region of actin, residues 1-28, contains a biologically important binding site for myosin.     

Identical behavior of the two active sites of myosin with respect to trinitrophenylation.

Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.