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. 1992 Nov;63(5):1306–1313. doi: 10.1016/S0006-3495(92)81717-4

Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.

B Hambly 1, K Franks 1, R Cooke 1
PMCID: PMC1261434  PMID: 1335782

Abstract

We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.

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Selected References

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  1. Arata T. Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers. J Mol Biol. 1990 Jul 20;214(2):471–478. doi: 10.1016/0022-2836(90)90194-Q. [DOI] [PubMed] [Google Scholar]
  2. Barnett V. A., Fajer P., Polnaszek C. F., Thomas D. D. High-Resolution Detection of muscle Crossbridge Orientation by Electron Paramagnetic Resonance. Biophys J. 1986 Jan;49(1):144–147. doi: 10.1016/S0006-3495(86)83628-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berger C. L., Svensson E. C., Thomas D. D. Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond rotational motions of myosin heads bound to actin. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8753–8757. doi: 10.1073/pnas.86.22.8753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borejdo J., Putnam S., Morales M. F. Fluctuations in polarized fluorescence: evidence that muscle cross bridges rotate repetitively during contraction. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6346–6350. doi: 10.1073/pnas.76.12.6346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burghardt T. P., Ando T., Borejdo J. Evidence for cross-bridge order in contraction of glycerinated skeletal muscle. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7515–7519. doi: 10.1073/pnas.80.24.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooke R., Crowder M. S., Thomas D. D. Orientation of spin labels attached to cross-bridges in contracting muscle fibres. Nature. 1982 Dec 23;300(5894):776–778. doi: 10.1038/300776a0. [DOI] [PubMed] [Google Scholar]
  7. Cooke R. The mechanism of muscle contraction. CRC Crit Rev Biochem. 1986;21(1):53–118. doi: 10.3109/10409238609113609. [DOI] [PubMed] [Google Scholar]
  8. Crowder M. S., Cooke R. Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers. Biophys J. 1987 Feb;51(2):323–333. doi: 10.1016/S0006-3495(87)83338-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crowder M. S., Cooke R. The effect of myosin sulphydryl modification on the mechanics of fibre contraction. J Muscle Res Cell Motil. 1984 Apr;5(2):131–146. doi: 10.1007/BF00712152. [DOI] [PubMed] [Google Scholar]
  10. Dos Remedios C. G., Millikan R. G., Morales M. F. Polarization of tryptophan fluorescence from single striated muscle fibers. A molecular probe of contractile state. J Gen Physiol. 1972 Jan;59(1):103–120. doi: 10.1085/jgp.59.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eisenberg E., Hill T. L., Chen Y. Cross-bridge model of muscle contraction. Quantitative analysis. Biophys J. 1980 Feb;29(2):195–227. doi: 10.1016/S0006-3495(80)85126-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fajer P. G., Fajer E. A., Matta J. J., Thomas D. D. Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high-resolution study with deuterated spin labels. Biochemistry. 1990 Jun 19;29(24):5865–5871. doi: 10.1021/bi00476a031. [DOI] [PubMed] [Google Scholar]
  13. Fajer P. G., Fajer E. A., Thomas D. D. Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5538–5542. doi: 10.1073/pnas.87.14.5538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geeves M. A. The dynamics of actin and myosin association and the crossbridge model of muscle contraction. Biochem J. 1991 Feb 15;274(Pt 1):1–14. doi: 10.1042/bj2740001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hambly B., Franks K., Cooke R. Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers. Biophys J. 1991 Jan;59(1):127–138. doi: 10.1016/S0006-3495(91)82205-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hibberd M. G., Dantzig J. A., Trentham D. R., Goldman Y. E. Phosphate release and force generation in skeletal muscle fibers. Science. 1985 Jun 14;228(4705):1317–1319. doi: 10.1126/science.3159090. [DOI] [PubMed] [Google Scholar]
  17. Hibberd M. G., Trentham D. R. Relationships between chemical and mechanical events during muscular contraction. Annu Rev Biophys Biophys Chem. 1986;15:119–161. doi: 10.1146/annurev.bb.15.060186.001003. [DOI] [PubMed] [Google Scholar]
  18. Hofmann P. A., Metzger J. M., Greaser M. L., Moss R. L. Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers. J Gen Physiol. 1990 Mar;95(3):477–498. doi: 10.1085/jgp.95.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huxley H. E., Faruqi A. R. Time-resolved X-ray diffraction studies on vertebrate striated muscle. Annu Rev Biophys Bioeng. 1983;12:381–417. doi: 10.1146/annurev.bb.12.060183.002121. [DOI] [PubMed] [Google Scholar]
  20. Katoh T., Lowey S. Mapping myosin light chains by immunoelectron microscopy. Use of anti-fluorescyl antibodies as structural probes. J Cell Biol. 1989 Oct;109(4 Pt 1):1549–1560. doi: 10.1083/jcb.109.4.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murray J. M., Weber A., Knox M. K. Myosin subfragment 1 binding to relaxed actin filaments and steric model of relaxation. Biochemistry. 1981 Feb 3;20(3):641–649. doi: 10.1021/bi00506a030. [DOI] [PubMed] [Google Scholar]
  22. Pate E., Cooke R. A model of crossbridge action: the effects of ATP, ADP and Pi. J Muscle Res Cell Motil. 1989 Jun;10(3):181–196. doi: 10.1007/BF01739809. [DOI] [PubMed] [Google Scholar]
  23. Pate E., Cooke R. Energetics of the actomyosin bond in the filament array of muscle fibers. Biophys J. 1988 Apr;53(4):561–573. doi: 10.1016/S0006-3495(88)83136-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reedy M. K., Holmes K. C., Tregear R. T. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature. 1965 Sep 18;207(5003):1276–1280. doi: 10.1038/2071276a0. [DOI] [PubMed] [Google Scholar]
  25. Sleep J., Glyn H. Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate. Biochemistry. 1986 Mar 11;25(5):1149–1154. doi: 10.1021/bi00353a030. [DOI] [PubMed] [Google Scholar]
  26. Sutoh K., Yamamoto K., Wakabayashi T. Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog. Proc Natl Acad Sci U S A. 1986 Jan;83(2):212–216. doi: 10.1073/pnas.83.2.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sutoh K., Yamamoto K., Wakabayashi T. Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system. J Mol Biol. 1984 Sep 15;178(2):323–339. doi: 10.1016/0022-2836(84)90147-5. [DOI] [PubMed] [Google Scholar]
  28. Svensson E. C., Thomas D. D. ATP induces microsecond rotational motions of myosin heads crosslinked to actin. Biophys J. 1986 Nov;50(5):999–1002. doi: 10.1016/S0006-3495(86)83541-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas D. D. Spectroscopic probes of muscle cross-bridge rotation. Annu Rev Physiol. 1987;49:691–709. doi: 10.1146/annurev.ph.49.030187.003355. [DOI] [PubMed] [Google Scholar]
  30. Titus M. A., Ashiba G., Szent-Györgyi A. G. SH-1 modification of rabbit myosin interferes with calcium regulation. J Muscle Res Cell Motil. 1989 Feb;10(1):25–33. doi: 10.1007/BF01739854. [DOI] [PubMed] [Google Scholar]
  31. Yanagida T. Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of epsilon-ATP, epsilon-ADP and epsilon-AMPPNP detected by polarized fluorescence. J Mol Biol. 1981 Mar 15;146(4):539–560. doi: 10.1016/0022-2836(81)90046-2. [DOI] [PubMed] [Google Scholar]

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