• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcinvestThe Journal of Clinical InvestigationCurrent IssueArchiveSubscriptionAbout the Journal
J Clin Invest. Mar 1, 1997; 99(5): 1010–1015.
PMCID: PMC507909

Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.


Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

Full Text

The Full Text of this article is available as a PDF (295K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Manstein DJ, Ruppel KM, Spudich JA. Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum. Science. 1989 Nov 3;246(4930):656–658. [PubMed]
  • Uyeda TQ, Spudich JA. A functional recombinant myosin II lacking a regulatory light chain-binding site. Science. 1993 Dec 17;262(5141):1867–1870. [PubMed]
  • Ruppel KM, Uyeda TQ, Spudich JA. Role of highly conserved lysine 130 of myosin motor domain. In vivo and in vitro characterization of site specifically mutated myosin. J Biol Chem. 1994 Jul 22;269(29):18773–18780. [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Howard PK, Ahern KG, Firtel RA. Establishment of a transient expression system for Dictyostelium discoideum. Nucleic Acids Res. 1988 Mar 25;16(6):2613–2623. [PMC free article] [PubMed]
  • Ruppel KM, Egelhoff TT, Spudich JA. Purification of a functional recombinant myosin fragment from Dictyostelium discoideum. Ann N Y Acad Sci. 1990;582:147–155. [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]
  • Miyata H, Hakozaki H, Yoshikawa H, Suzuki N, Kinosita K, Jr, Nishizaka T, Ishiwata S. Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay. J Biochem. 1994 Apr;115(4):644–647. [PubMed]
  • Svoboda K, Mitra PP, Block SM. Fluctuation analysis of motor protein movement and single enzyme kinetics. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11782–11786. [PMC free article] [PubMed]
  • Kron SJ, Spudich JA. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6272–6276. [PMC free article] [PubMed]
  • Kodama T, Fukui K, Kometani K. The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate. J Biochem. 1986 May;99(5):1465–1472. [PubMed]
  • Smith CA, Rayment I. X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. Biochemistry. 1996 Apr 30;35(17):5404–5417. [PubMed]
  • Lee C. Predicting protein mutant energetics by self-consistent ensemble optimization. J Mol Biol. 1994 Feb 25;236(3):918–939. [PubMed]
  • Uyeda TQ, Kron SJ, Spudich JA. Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin. J Mol Biol. 1990 Aug 5;214(3):699–710. [PubMed]
  • Milligan RA. Protein-protein interactions in the rigor actomyosin complex. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):21–26. [PMC free article] [PubMed]
  • Sweeney HL, Straceski AJ, Leinwand LA, Tikunov BA, Faust L. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J Biol Chem. 1994 Jan 21;269(3):1603–1605. [PubMed]
  • Whittaker M, Wilson-Kubalek EM, Smith JE, Faust L, Milligan RA, Sweeney HL. A 35-A movement of smooth muscle myosin on ADP release. Nature. 1995 Dec 14;378(6558):748–751. [PubMed]
  • Uyeda TQ, Abramson PD, Spudich JA. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4459–4464. [PMC free article] [PubMed]
  • Poetter K, Jiang H, Hassanzadeh S, Master SR, Chang A, Dalakas MC, Rayment I, Sellers JR, Fananapazir L, Epstein ND. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet. 1996 May;13(1):63–69. [PubMed]
  • Lankford EB, Epstein ND, Fananapazir L, Sweeney HL. Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J Clin Invest. 1995 Mar;95(3):1409–1414. [PMC free article] [PubMed]
  • Sutoh K, Ando M, Sutoh K, Toyoshima YY. Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7711–7714. [PMC free article] [PubMed]
  • Johara M, Toyoshima YY, Ishijima A, Kojima H, Yanagida T, Sutoh K. Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2127–2131. [PMC free article] [PubMed]
  • Duong AM, Reisler E. C-terminus on actin: spectroscopic and immunochemical examination of its role in actomyosin interactions. Adv Exp Med Biol. 1994;358:59–70. [PubMed]
  • Miller CJ, Reisler E. Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin. Biochemistry. 1995 Feb 28;34(8):2694–2700. [PubMed]
  • Miller CJ, Cheung P, White P, Reisler E. Actin's view of actomyosin interface. Biophys J. 1995 Apr;68(4 Suppl):50S–54S. [PMC free article] [PubMed]
  • Tanigawa G, Jarcho JA, Kass S, Solomon SD, Vosberg HP, Seidman JG, Seidman CE. A molecular basis for familial hypertrophic cardiomyopathy: an alpha/beta cardiac myosin heavy chain hybrid gene. Cell. 1990 Sep 7;62(5):991–998. [PubMed]
  • Geisterfer-Lowrance AA, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, Seidman JG. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell. 1990 Sep 7;62(5):999–1006. [PubMed]
  • Seidman CE, Seidman JG. Mutations in cardiac myosin heavy chain genes cause familial hypertrophic cardiomyopathy. Mol Biol Med. 1991 Apr;8(2):159–166. [PubMed]
  • Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3;77(5):701–712. [PubMed]
  • Watkins H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, Maron BJ, Seidman JG, Seidman CE. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec;11(4):434–437. [PubMed]
  • Watkins H, McKenna WJ, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, Spirito P, Matsumori A, Moravec CS, Seidman JG, et al. Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med. 1995 Apr 20;332(16):1058–1064. [PubMed]
  • Watkins H, MacRae C, Thierfelder L, Chou YH, Frenneaux M, McKenna W, Seidman JG, Seidman CE. A disease locus for familial hypertrophic cardiomyopathy maps to chromosome 1q3. Nat Genet. 1993 Apr;3(4):333–337. [PubMed]
  • Bonne G, Carrier L, Bercovici J, Cruaud C, Richard P, Hainque B, Gautel M, Labeit S, James M, Beckmann J, et al. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec;11(4):438–440. [PubMed]
  • Rayment I, Holden HM, Sellers JR, Fananapazir L, Epstein ND. Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3864–3868. [PMC free article] [PubMed]
  • Anan R, Greve G, Thierfelder L, Watkins H, McKenna WJ, Solomon S, Vecchio C, Shono H, Nakao S, Tanaka H, et al. Prognostic implications of novel beta cardiac myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy. J Clin Invest. 1994 Jan;93(1):280–285. [PMC free article] [PubMed]
  • Fisher AJ, Smith CA, Thoden JB, Smith R, Sutoh K, Holden HM, Rayment I. X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. Biochemistry. 1995 Jul 18;34(28):8960–8972. [PubMed]
  • Sablin EP, Kull FJ, Cooke R, Vale RD, Fletterick RJ. Crystal structure of the motor domain of the kinesin-related motor ncd. Nature. 1996 Apr 11;380(6574):555–559. [PubMed]
  • Kull FJ, Sablin EP, Lau R, Fletterick RJ, Vale RD. Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature. 1996 Apr 11;380(6574):550–555. [PMC free article] [PubMed]
  • Consevage MW, Salada GC, Baylen BG, Ladda RL, Rogan PK. A new missense mutation, Arg719Gln, in the beta-cardiac heavy chain myosin gene of patients with familial hypertrophic cardiomyopathy. Hum Mol Genet. 1994 Jun;3(6):1025–1026. [PubMed]
  • Watkins H, Thierfelder L, Anan R, Jarcho J, Matsumori A, McKenna W, Seidman JG, Seidman CE. Independent origin of identical beta cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy. Am J Hum Genet. 1993 Dec;53(6):1180–1185. [PMC free article] [PubMed]
  • Manstein DJ, Titus MA, De Lozanne A, Spudich JA. Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 1989 Mar;8(3):923–932. [PMC free article] [PubMed]

Articles from The Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...