Summary
We have performed electron paramagnetic resonance (e.p.r.) experiments on spin-labelled myosin heads in glycerinated insect flight muscle fibres and myofibrils in rigor. Conventional e.p.r. was used to determine the orientation distribution of spin labels relative to the fibre axis, and saturation transfer e.p.r. was used to determine the submillisecond rotational mobility. An iodoacetamide spin label has previously been shown to react selectively with a reactive SH group on the myosin heads of rabbit skeletal fibres, and this label appeared to react with a similar group in insect fibres. Although selective labelling of this group was achieved in insect fibres and myofibrils, the reaction proceeded more slowly than in skeletal muscle, making it more difficult to label myosin heads selectively in insect muscle. The fraction of spin labels bound to myosin was 0.88±0.07 in insect fibres and 0.97±0.05 in rabbit. The fraction of myosin heads labelled was 0.65±0.15 for insect and 0.81±0.10 for rabbit. Both conventional and saturation transfer e.p.r. spectra of insect myosin, myofibrils and fibres were very similar to those of rabbit. The orientation distribution of spin labels relative to the fibre axis in rigor was narrow (16° for rabbit, 22° for insect), and the centre of the angular distribution was essentially the same for insect as for rabbit (68°–69°). This high degree of orientation was accompanied by strong immobilization of the probe on the microsecond time scale. The same immobilization was observed for rigor myofibrils as for purified myosin in the presence of excess actin, but considerable microsecond rotational motion was observed in myosin filaments free of actin. Thus, in insect as well as rabbit, assuming that the labelled heads are representative of all heads, more than 80% of the myosin heads appear to bind to actin in rigor, and the actomyosin bonds are rotationally rigid and oriented within a narrow angular range with respect to the fibre axis.
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References
BARNETT, V. A. & THOMAS, D. D. (1983) Saturation transfer e.p.r. on spin-labelled muscle fibers: dependence on sarcomere length.J. molec. Biol. (in press).
BOREJDO, J. & PUTNAM, S. (1977) Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation.Biochim. Biophys. Acta 459, 578–95.
BULLARD, B. & REEDY, M. K. (1973) How many myosins per cross-bridge.Cold Spring Harb. Symp. quant. Biol. 37, 423–8.
CLARKE, M. & TREGEAR, R. T. (1980) Supercooling as a method for storing glycerol-extracted muscle fibers.J. Physiol. 308, 104–5.
COOKE, R. & BIALEK, W. (1979) Contraction of glycerinated muscle fibres as a function of the ATP concentration.Biophys. J. 28, 241–58.
COOKE, R. & FRANKS, K. (1980) All myosin heads form bonds with actin in rigor rabbit skeletal muscle.Biochemistry 19, 2265–9.
DUKE, J., TAKASHI, R., UE, K. & MORALES, M. (1976) Reciprocal reactivities of specific thiols when actin binds to myosin.Proc. natn. Acad. Sci. 73, 302–6.
GRACEFFA, P. & SEIDEL, J. C. (1980) A reaction involving protein sulfhydryl groups, bound spin label, and K3Fe(CN)6 as a probe of sulfhydryl proximity in myosin and actomyosin.Biochemistry 19, 33–9.
HASELGROVE, J. C. & REEDY, M. K. (1978) Modeling rigor cross-bridge patterns in muscle.Biophys. J. 24, 713–28.
HOLMES, K. C., TREGEAR, R. T. & BARRINGTON, L. J. (1980) Interpretation of the low angle X-ray diffraction from insect flight muscle in rigor.Proc. R. Soc. 207, 13–33.
HUXLEY, A. F. & SIMMONS, R. M. (1971) Proposed mechanism of force generation in striated muscle.Nature 233, 533–8.
LOVELL, S. J. & HARRINGTON, W. F. (1981) Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor.J. molec. Biol. 149, 659–74.
LOVELL, S. J., KNIGHT, P. J. & HARRINGTON, W. F. (1981) Fraction of myosin heads bound to thin filaments in rigor fibrils from insect flight and vertebrate muscles.Nature 293, 664–6.
MILLER, A. & TREGEAR, R. T. (1972) Structure of insect fibrillar flight muscle: the presence and absence of ATP.J. molec. Biol. 70, 85–104.
OFFER, G., COUCH, J., O'BRIEN, E. & ELLIOTT, A. (1981) Arrangement of crossbridges in insect flight muscle in rigor,J. molec. Biol. 151, 663–702.
OFFER, G. & ELLIOT, A. (1978) Can a myosin molecule bind two actin filaments?Nature 271, 325–9.
PRINGLE, J. W. S. (1967) The contractile mechanism of insect fibrillar muscle.Prog. Biophys. molec. Biol. 17, 1–60.
REEDY, M. K. (1967) Crossbridges and periods in insect flight muscle.Am. Zool. 7, 465–81.
REEDY, M. K. (1968) Ultrasound of insect flight muscle.J. molec. Biol. 31, 155–76.
REEDY, M. K., HOLMES, K. C. & TREGEAR, R. T. (1965) Induced changes in orientation of the crossbridges of glycerinated insect flight muscle.Nature 207, 1276–80.
REEDY, M. K., LEONARD, K. R., FREEMAN, R. & ARAD, J. (1981) Thick filament mass determination by electron scattering measurements with the scanning transmission electron microscope.J. Musc. Res. Cell Motility 2, 45–64.
SEIDEL, J. C., CHOPEK, M. & GERGELY, J. (1970) Effect of nucleotides and pyrophosphate on spin labels bound to S1 thiol groups of myosin.Biochemistry 9, 3265.
SQUIRE, J. M. (1971) General model of myosin filament structure.J. molec. Biol. 72, 125–38.
SQUIRE, J. M. (1977) The structure of insect thick filaments. InInsect Flight Muscle (edited by TREGEAR, R. T.) pp. 91–112. Amsterdam: North Holland.
THOMAS, D. D. (1978) Large-scale rotational motions detected by EPR and fluorescence.Biophys. J. 24, 439–62.
THOMAS, D. D. & COOKE, R. (1980) Orientation of spin-labelled myosin heads in glycerinated muscle.Biophys. J. 32, 891–906.
THOMAS, D. D., DALTON, L. R. & HYDE, J. S. (1976) Rotational diffusion studied by passage saturation transfer electron paramagnetic resonance.J. chem. Phys. 65, 3006–24.
THOMAS, D. D., ISHIWATA, S., SEIDEL, J. C. & GERGELY, J. (1980) Submillisecond rotational dynamics of spin-labeled cross-bridges in myofibrils.Biophys. J. 32, 873–90.
THOMAS, D. D., LINDAHL, K., WENDT, C. & BARNETT, V. A. (1983) Theoretical analysis of EPR spectra from oriented muscle fibres.Biophys. J. (in press).
THOMAS, D. D., SEIDEL, J. C., HYDE, J. S. & GERGELY, J. (1975) Motion of S-1 in myosin and its supramolecular complexes: saturation transfer electron paramagnetic resonance.Proc. natn. Acad. Sci. 72, 1729–33.
TREGEAR, R. T. & MARSTON, S. B. (1979) The crossbridge theory.Ann. Rev. Physiol. 41, 723–36.
WRAY, J. S. (1979) Filament geometry and the activation of insect flight muscles.Nature 280, 325–7.
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Thomas, D.D., Cooke, R. & Barnett, V.A. Orientation and rotational mobility of spin-labelled myosin heads in insect flight muscle in rigor. J Muscle Res Cell Motil 4, 367–378 (1983). https://doi.org/10.1007/BF00712002
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DOI: https://doi.org/10.1007/BF00712002