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J Cell Biol. 1989 August 1; 109(2): 529–538.
PMCID: PMC2115714

Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP

Abstract

We have used electron microscopy and proteolytic susceptibility to study the structural basis of myosin-linked regulation in synthetic filaments of scallop striated muscle myosin. Using papain as a probe of the structure of the head-rod junction, we find that this region of myosin is approximately five times more susceptible to proteolytic attack under activating (ATP/high Ca2+) or rigor (no ATP) conditions than under relaxing conditions (ATP/low Ca2+). A similar result was obtained with native myosin filaments in a crude homogenate of scallop muscle. Proteolytic susceptibility under conditions in which ADP or adenosine 5'-(beta, gamma-imidotriphosphate) (AMPPNP) replaced ATP was similar to that in the absence of nucleotide. Synthetic myosin filaments negatively stained under relaxing conditions showed a compact structure, in which the myosin cross-bridges were close to the filament backbone and well ordered, with a clear 14.5-nm axial repeat. Under activating or rigor conditions, the cross-bridges became clumped and disordered and frequently projected further from the filament backbone, as has been found with native filaments; when ADP or AMPPNP replaced ATP, the cross-bridges were also disordered. We conclude (a) that Ca2+ and ATP affect the affinity of the myosin cross-bridges for the filament backbone or for each other; (b) that the changes observed in the myosin filaments reflect a property of the myosin molecules alone, and are unlikely to be an artifact of negative staining; and (c) that the ordered structure occurs only in the relaxed state, requiring both the presence of hydrolyzed ATP on the myosin heads and the absence of Ca2+.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adelstein RS, Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. [PubMed]
  • Bennett AJ, Patel N, Wells C, Bagshaw CR. 8-Anilino-1-naphthalenesulphonate, a fluorescent probe for the regulatory light chain binding site of scallop myosin. J Muscle Res Cell Motil. 1984 Apr;5(2):165–182. [PubMed]
  • Caspar DL, Cohen C, Longley W. Tropomyosin: crystal structure, polymorphism and molecular interactions. J Mol Biol. 1969 Apr 14;41(1):87–107. [PubMed]
  • Chalovich JM, Chantler PD, Szent-Gyorgyi AG, Eisenberg E. Regulation of molluscan actomyosin ATPase activity. J Biol Chem. 1984 Feb 25;259(4):2617–2621. [PMC free article] [PubMed]
  • Chantler PD, Szent-Györgyi AG. Spectroscopic studies on invertebrate myosins and light chains. Biochemistry. 1978 Dec 12;17(25):5440–5448. [PubMed]
  • Clarke ML, Hofman W, Wray JS. ATP binding and crossbridge structure in muscle. J Mol Biol. 1986 Oct 5;191(3):581–585. [PubMed]
  • Collins JH, Jakes R, Kendrick-Jones J, Leszyk J, Barouch W, Theibert JL, Spiegel J, Szent-Györgyi AG. Amino acid sequence of myosin essential light chain from the scallop Aquipecten irradians. Biochemistry. 1986 Nov 18;25(23):7651–7656. [PubMed]
  • Craig R, Padrón R, Kendrick-Jones J. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments. J Cell Biol. 1987 Sep;105(3):1319–1327. [PMC free article] [PubMed]
  • Craig R, Smith R, Kendrick-Jones J. Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. Nature. 302(5907):436–439. [PubMed]
  • Craig R, Szent-Györgyi AG, Beese L, Flicker P, Vibert P, Cohen C. Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin. J Mol Biol. 1980 Jun 15;140(1):35–55. [PubMed]
  • Crowther RA, Padrón R, Craig R. Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle. J Mol Biol. 1985 Aug 5;184(3):429–439. [PubMed]
  • Hardwicke PM, Szent-Györgyi AG. Proximity of regulatory light chains in scallop myosin. J Mol Biol. 1985 May 25;183(2):203–211. [PubMed]
  • Hardwicke PM, Wallimann T, Szent-Györgyi AG. Regulatory and essential light-chain interactions in scallop myosin. I. Protection of essential light-chain thiol groups by regulatory light-chains. J Mol Biol. 1982 Mar 25;156(1):141–152. [PubMed]
  • Hardwicke PM, Wallimann T, Szent-Györgyi AG. Light-chain movement and regulation in scallop myosin. Nature. 1983 Feb 10;301(5900):478–482. [PubMed]
  • Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 1979 May;81(2):275–300. [PMC free article] [PubMed]
  • Hinssen H, D'Haese J, Small JV, Sobieszek A. Mode of filament assembly of myosins from muscle and nonmuscle cells. J Ultrastruct Res. 1978 Sep;64(3):282–302. [PubMed]
  • Ikebe M, Hartshorne DJ. Conformation-dependent proteolysis of smooth-muscle myosin. J Biol Chem. 1984 Oct 10;259(19):11639–11642. [PubMed]
  • Ikebe M, Hartshorne DJ. Proteolysis and actin-binding properties of 10S and 6S smooth muscle myosin: identification of a site protected from proteolysis in the 10S conformation and by the binding of actin. Biochemistry. 1986 Oct 7;25(20):6177–6185. [PubMed]
  • Ikebe M, Ogihara S. Phosphorylation-dependent and ATP-induced changes in structural array in gizzard myosin filament bundles. J Biochem. 1982 Dec;92(6):1973–1977. [PubMed]
  • Kendrick-Jones J, Lehman W, Szent-Györgyi AG. Regulation in molluscan muscles. J Mol Biol. 1970 Dec 14;54(2):313–326. [PubMed]
  • Kensler RW, Levine RJ. An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments. J Cell Biol. 1982 Feb;92(2):443–451. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lehman W, Szent-Györgyi AG. Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom. J Gen Physiol. 1975 Jul;66(1):1–30. [PMC free article] [PubMed]
  • Levine RJ, Chantler PD, Kensler RW. Arrangement of myosin heads on Limulus thick filaments. J Cell Biol. 1988 Nov;107(5):1739–1747. [PMC free article] [PubMed]
  • Lowey S, Slayter HS, Weeds AG, Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Margossian SS, Stafford WF, 3rd, Lowey S. Homogeneity of myosin subfragments by equilibrium centrifugation. Biochemistry. 1981 Apr 14;20(8):2151–2155. [PubMed]
  • Moos C, Offer G, Starr R, Bennett P. Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol. 1975 Sep 5;97(1):1–9. [PubMed]
  • Okamoto Y, Sekine T. A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide. J Biol Chem. 1987 Jun 15;262(17):7951–7954. [PubMed]
  • Onishi H, Watanabe S. Correlation between the papain digestibility and the conformation of 10s-myosin from chicken gizzard. J Biochem. 1984 Mar;95(3):899–902. [PubMed]
  • Padrón R, Huxley HE. The effect of the ATP analogue AMPPNP on the structure of crossbridges in vertebrate skeletal muscles: X-ray diffraction and mechanical studies. J Muscle Res Cell Motil. 1984 Dec;5(6):613–655. [PubMed]
  • Padrón R, Alamo L, Craig R, Caputo C. A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension. J Microsc. 1988 Aug;151(Pt 2):81–102. [PubMed]
  • Pardee JD, Spudich JA. Purification of muscle actin. Methods Enzymol. 1982;85(Pt B):164–181. [PubMed]
  • Pinset-Härström I. MgATP specifically controls in vitro self-assembly of vertebrate skeletal myosin in the physiological pH range. J Mol Biol. 1985 Mar 5;182(1):159–172. [PubMed]
  • Pinset-Härström I, Truffy J. Effect of adenosine triphosphate, inorganic phosphate and divalent cations on the size and structure of synthetic myosin filaments. An electron microscope study. J Mol Biol. 1979 Oct 15;134(1):173–188. [PubMed]
  • Pollard TD. Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation. J Cell Biol. 1975 Oct;67(1):93–104. [PMC free article] [PubMed]
  • Salmon ED, DeRosier D. A surveying optical diffractometer. J Microsc. 1981 Sep;123(Pt 3):239–247. [PubMed]
  • Schacterle GR, Pollack RL. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. [PubMed]
  • Sellers JR. Phosphorylation-dependent regulation of Limulus myosin. J Biol Chem. 1981 Sep 10;256(17):9274–9278. [PubMed]
  • Sherry JM, Górecka A, Aksoy MO, Dabrowska R, Hartshorne DJ. Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. Biochemistry. 1978 Oct 17;17(21):4411–4418. [PubMed]
  • Spudich JA, Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed]
  • Stafford WF, 3rd, Szentkiralyi EM, Szent-Györgyi AG. Regulatory properties of single-headed fragments of scallop myosin. Biochemistry. 1979 Nov 27;18(24):5273–5280. [PubMed]
  • Suzuki H, Kondo Y, Carlos AD, Seidel JC. Effects of phosphorylation, MgATP, and ionic strength on the rates of papain degradation of heavy and light chains of smooth muscle heavy meromyosin at the S1-S2 junction. J Biol Chem. 1988 Aug 5;263(22):10974–10979. [PubMed]
  • Suzuki H, Stafford WF, 3rd, Slayter HS, Seidel JC. A conformational transition in gizzard heavy meromyosin involving the head-tail junction, resulting in changes in sedimentation coefficient, ATPase activity, and orientation of heads. J Biol Chem. 1985 Nov 25;260(27):14810–14817. [PubMed]
  • Szent-Györgyi AG, Szentkiralyi EM, Kendrick-Jonas J. The light chains of scallop myosin as regulatory subunits. J Mol Biol. 1973 Feb 25;74(2):179–203. [PubMed]
  • Szentkiralyi EM. Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide. J Muscle Res Cell Motil. 1984 Apr;5(2):147–164. [PubMed]
  • TAUSSKY HH, SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed]
  • Trybus KM, Lowey S. Conformational states of smooth muscle myosin. Effects of light chain phosphorylation and ionic strength. J Biol Chem. 1984 Jul 10;259(13):8564–8571. [PubMed]
  • Vibert P, Castellani L. Substructure and accessory proteins in scallop myosin filaments. J Cell Biol. 1989 Aug;109(2):539–547. [PMC free article] [PubMed]
  • Vibert P, Cohen C. Domains, motions and regulation in the myosin head. J Muscle Res Cell Motil. 1988 Aug;9(4):296–305. [PubMed]
  • Vibert P, Craig R. Electron microscopy and image analysis of myosin filaments from scallop striated muscle. J Mol Biol. 1983 Apr 5;165(2):303–320. [PubMed]
  • Vibert P, Craig R. Structural changes that occur in scallop myosin filaments upon activation. J Cell Biol. 1985 Sep;101(3):830–837. [PMC free article] [PubMed]
  • Wells C, Bagshaw CR. Calcium regulation of molluscan myosin ATPase in the absence of actin. Nature. 1985 Feb 21;313(6004):696–697. [PubMed]
  • Wells C, Warriner KE, Bagshaw CR. Fluorescence studies on the nucleotide- and Ca2+-binding domains of molluscan myosin. Biochem J. 1985 Oct 1;231(1):31–38. [PubMed]
  • White HD. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol. 1982;85(Pt B):698–708. [PubMed]
  • Winkelmann DA, Almeda S, Vibert P, Cohen C. A new myosin fragment: visualization of the regulatory domain. Nature. 1984 Feb 23;307(5953):758–760. [PubMed]

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