Smooth Muscle Heavy Meromyosin Research Articles

Novel ADP State Found in Smooth Muscle Heavy Meromyosin by CryoEM.

Novel ADP State Found in Smooth Muscle Heavy Meromyosin by CryoEM.

Cryoem Structure Determination of Actin Decorated with Smooth Muscle Heavy Meromyosin

Cryoem Structure Determination of Actin Decorated with Smooth Muscle Heavy Meromyosin

Constitutive phosphorylation of myosin phosphatase targeting subunit-1 in smooth muscle.

Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wild-type or MYPT1-deficient tissues. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to inhibit MLCP activity. In summary, MYPT1 deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed.

Atomic-Level Visualization of Smooth Muscle Activation by Phosphorylation of the Myosin Regulatory Light Chain

We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC's N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions between the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the RLC core and the dynamic PD. Smooth muscle RLC assumes two structural states in both states of activation: phosphorylation shifts the equilibrium of these two structural states from primarily occupying the compact closed state to favoring the extended open state of the PD, relative to the RLC core. Compared to single-headed S1 (Kast et al., PNAS., 2010), the open state RLC conformation of unphosphorylated HMM is more compact, presumably due to head-head or RLC-RLC interactions. All-atom MD simulations corroborate our TR-FRET studies, and reveal specific salt-bridges that stabilize each structural state. The hypotheses generated from our MD simulations are being tested experimentally by TR-FRET via site-directed mutagenesis to manipulate charge. These studies offer the first atomic-resolution insight into the structural dynamics of RLC phosphorylation, and a similar approach should be applicable to striated muscle. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).

Function of the head–tail junction in the activity of myosin II

All class II myosins have the conserved amino acid sequence Pro-Leu-Leu at their head–tail junctions. We systematically altered this sequence in smooth muscle heavy meromyosin (HMM) by site-directed mutagenesis and examined the effects of these mutations on actin–myosin interactions. Deletion of the proline and second leucine did not cause any noticeable change in either actin-activated ATPase activity or actin-sliding velocity. In contrast, deletion of the two leucine residues and substitution of the first leucine with alanine resulted in a 14-fold and 5-fold decrease, respectively, in actin-activated ATPase activity. However, both these mutations did not appreciably affect actin-sliding velocity, which was consistent with a result that there was no considerable change in the ADP release rate from acto-HMM in the deletion mutant. In contrast to double-headed HMM, a single-headed subfragment-1 (S1) with a Leu-Leu deletion mutation exhibited actin activated ATPase activity similar to that by wild type S1. Our results suggest that the first leucine of the conserved Leu-Leu sequence at the head–tail junction profoundly affects the cooperativity between the two heads involved in the actin activated ATPase activity of myosin II.

Hydrogen-Deuterium Exchange Mass Spectrometry of Phosphorylation Induced Changes in Smooth Muscle Myosin

Inter-molecular interactions between two smooth muscle myosin heads have been observed using chemical cross linking and visualized by electron microscopy. In order to probe the nature of the phosphorylation-dependent head-head interactions, we performed Hydrogen-Deuterium exchange electrospray ionization mass spectrometry (HDX-MS) on phosphorylated and unphosphorylated smooth muscle heavy mero myosin (HMM) from chicken gizzard. HDX rates are determined by solvent accesibility and the strength of hydrogen bonding and thus are a good fingerprint of intra- and intermolecular structural changes. The observed decrease in deuterium uptake upon phosphorylation in the coiled-coil and the hinge region of the heavy chain (residues 864-877), is consistent with the unraveling of the myosin dimer at the hinge. Unexpectedly, we have observed phosphorylation dependent changes in the regulatory light chain (RLC 58-69), which are not implicated in the RLC-RLC or RLC-heavy chain interface. No changes were observed for N-terminal region of RLC that was postulated to undergo major secondary structure changes, or in the heavy chain actin binding site (residues 397-437), or any of the residues that form the head-head interface in the EM model of Wendt & Taylor. This latter finding indicates that the interaction between the heads is probably transient in solution, or in the unlikely case, interacting heads constitute a very small fraction of total protein.

Modification of Interface between Regulatory and Essential Light Chains Hampers Phosphorylation-dependent Activation of Smooth Muscle Myosin

We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, V(max), and K(ATPase) of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca(2+) binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218-8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.

Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation: Implications for the Structure of Myosin with One Head Phosphorylated

Smooth muscle myosin (smM) and heavy meromyosin (smHMM) are activated by regulatory light chain (RLC) phosphorylation but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions involving motor domains and the essential light chain (ELC) [Wendt et al., PNAS 98: 4361 (2001)]. The “free head” can bind to actin, but the actin-binding interface of the “blocked head” is involved in interactions with the free head. We report here a 3-D structure for phosphorylated, active smHMM obtained using electron crystallography of 2-D arrays, and an atomic model obtained by fitting using normal mode flexible fitting. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state, but occur between separate molecules. The interface between heads of phosphorylated smHMM is less extensive and somewhat altered in orientation compared with that of dephosphorylated smHMM. The light chain binding domain of phosphorylated and several dephosphorylated myosin structures show systematic differences. However, the major difference appears to be the relationship between the motor domain and the ELC in a phosphorylated head compared to that of the “blocked head” of dephosphorylated smHMM. We hypothesize that RLC phosphorylation disrupts the inhibited conformation primarily by its effect on the “blocked head” rather than the “free head”. Singly phosphorylated smHMM is not compatible with the closed conformation if the “blocked head” is phosphorylated. The implications of this observation for myosin activation at low levels of phosphorylation in smooth muscle will be discussed. Supported by grants from the NIAMS, NHLBI and NSF-MCB.

Phosphorylated Smooth Muscle Heavy Meromyosin Shows an Open Conformation Linked to Activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head–head interactions between motor domains. The “free head” can bind to actin, but the actin binding interface of the “blocked head” is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head–head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the “blocked” head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion

Smooth muscle myosin is activated by regulatory light chain (RLC) phosphorylation. In the unphosphorylated state the activity of both heads is suppressed due to an asymmetric, intramolecular interaction between the heads. The properties of myosin with only one of its two RLCs phosphorylated, a state likely to be present both during the activation and the relaxation phase of smooth muscle, is less certain despite much investigation. Here we further characterize the mechanical properties of an expressed heavy meromyosin (HMM) construct with only one of its RLCs phosphorylated (HMM-1P). This construct was previously shown to have more than 50% of the ATPase activity of fully phosphorylated myosin (HMM-2P) and to move actin at the same speed in a motility assay as HMM-2P (Rovner, A. S., Fagnant, P. M., and Trybus, K. M. (2006) Biochemistry 45, 5280-5289). Here we show that the unitary step size and attachment time to actin of HMM-1P is indistinguishable from that of HMM-2P. Force-velocity measurements on small ensembles show that HMM-1P can generate approximately half the force of HMM-2P, which may relate to the observed duty ratio of HMM-1P being approximately half that of HMM-2P. Therefore, single-phosphorylated smooth muscle HMM molecules are active species, and the head associated with the unphosphorylated RLC is mechanically competent, allowing it to make a substantial contribution to both motion and force generation during smooth muscle contraction.

The myosin C-loop is an allosteric actin contact sensor in actomyosin.

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

The Ups and Downs of Smooth Muscle Myosin Regulation

Since discovery that regulatory light chain (RLC) phosphorylation was the primary method of regulation of smooth and non-muscle myosin II, the structure of the phosphorylated and dephosphorylated forms has been a goal only partially realized. Regulation in both smooth muscle and the related scallop striated muscle myosin requires a 2-headed myosin species; single headed species are unregulated. Thus head to head interactions are key to achieving the inhibited state. A folded conformation of full length myosin, generally referred to as the 10S conformation was discovered early by conventional electron microscopy and in 1999, a structural explanation for the head-head interactions was obtained from 2-D arrays of dephosphorylated smooth muscle heavy meromyosin formed on lipid monolayers and imaged in 3-D by cryoEM. The structure was later confirmed by 2-D arrays of the 10S conformation of whole myosin. While this structure explained inhibition of the solubilized form of regulated myosins, it had not been observed in filaments. Surprisingly, the first observation of smooth muscle myosin-like head-head interactions in a thick filament was obtained from tarantula striated muscle, not from smooth muscle myosin. Even more surprising was the observation of a similar conformation in relaxed cardiac muscle thick filaments. Thus, these head-head interactions observed first in smooth muscle HMM, appear to be ubiquitous in relaxed muscle although still not confirmed for thick filaments in relaxed smooth muscle. Still to be determined is an explanation of the factors that can lead to solubilization and the location of the N-terminus of the RLC, whose location and structure has yet to be revealed, in the phosphorylated and dephosphorylated state. Theoretical modeling has provided possible explanations for several factors that affect regulation but has not yet yielded a coherent theory. Supported by NIAMSD.

H2O2-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin: CORRELATION TO EFFECTS OF H2O2 ON AIRWAY SMOOTH MUSCLE

The effect of H(2)O(2) on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P(i) release were measured by single-turnover kinetics. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.

1P158 リン酸化にともなう平滑筋ヘビメロミオシンの構造変化(筋肉(筋蛋白質・収縮),ポスター発表,第45回日本生物物理学会年会)

1P158 リン酸化にともなう平滑筋ヘビメロミオシンの構造変化(筋肉(筋蛋白質・収縮),ポスター発表,第45回日本生物物理学会年会)

A Unique ATP Hydrolysis Mechanism of Single-headed Processive Myosin, Myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.

Loop 1 of Transducer Region in Mammalian Class I Myosin, Myo1b, Modulates Actin Affinity, ATPase Activity, and Nucleotide Access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Asymmetric Photocross-Linking of Singly Phosphorylated Smooth Muscle Heavy Meromyosin

Although activities of smooth muscle myosin are regulated by phosphorylation, the molecular mechanisms of regulation have not been fully established. Phosphorylation of both heads of myosin is known to activate ATPase and motor activities, but the effects of phosphorylation of only one of the heads have not been established. Such information on singly phosphorylated myosin can serve to elucidate the molecular mechanism of the phosphorylation-dependent regulation. To understand the structural properties of the singly phosphorylated state, we prepared singly phosphorylated heavy meromyosin (HMM) containing a photoreactive benzophenone-labeled RLC and examined its photocross-linking reactivity. The two heads in the singly phosphorylated HMM showed different reactivities. The dephosphorylated RLC in the singly phosphorylated HMM was cross-linked to a heavy chain, like that in the dephosphorylated HMM, whereas the phosphorylated RLC did not react, like that in the fully phosphorylated HMM. These results indicate that the two heads of the singly phosphorylated HMM have an asymmetric structure, suggesting that phosphorylation of one head can to some extent activate smooth muscle HMM.

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.

1P175 平滑筋ヘビメロミオシンの構造変化のX線溶液散乱解析(筋肉 : 筋蛋白・収縮)

1P175 平滑筋ヘビメロミオシンの構造変化のX線溶液散乱解析(筋肉 : 筋蛋白・収縮)