Axial disposition of myosin heads in isometrically contracting muslces

ABSTRACT Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time.

INTRODUCTION

The meridional part of the x-ray diffraction diagram of striated muscles is proportional to the square of the modulus of the Fourier transform of the mass projection of the structure onto the muscle axis. Therefore, and especially given the elongated shape of the S1 sub-fragment (Rayment et al., 1993), the meridional reflections on the myosin layer lines, following a sequence of orders of a repeat of ~43.0 nm at rest, are sensitive to the axial orientation of the myosin heads. For these reasons, the myosin meridional reflections, and in particular the strongest order on the third myosin layer line (3M) at a rest spacing of 14.34 nm, have been extensively used as markers for myosin head disposition during various forms of contraction, initially in whole muscles (Huxley et al., 1981, 1982, 1983) and more recently in muscle fibers (Irving et al., 1992; Dobbie et al., 1998). These experiments have been interpreted as providing supportive, but not conclusive, evidence for models of contraction based on axial swings of various parts of the myosin molecule coupled to the hydrolysis of ATP (Huxley, 1969, 1974; Holmes, 1997). Because the knowledge of the phases associated with the diffraction features have been up to date lacking, the interpretation of the data had to rely on modeling. The fact that there is a pair of heads in the doubleheaded myosin molecule adds an additional complication, and generally, data were interpreted assuming that both heads in the pair had the same axial orientation.

Here we present experimental results where the meridional diffraction features at the plateau of isometric tension (P^sub o^) are recorded with the kind of angular resolution only achievable with third-generation synchrotron radiation (SR) sources. The data show that all the meridional reflections on the myosin layer lines, up to the 15th order (i.e., the 3M, 6M, 9M, and 15M, with the 12M being too weak to measure) are carved up by interference effects so that clusters of peaks appear on the meridional reflections due to the axial disposition of the heads. We show that with appropriate theoretical analysis of these effects one can extract phase information from which the axial distribution of the myosin heads can be determined. The results show that at Po, each myosin head in the pair has a distinctly different axial orientation.

MATERIALS AND METHODS

Data collection and reduction

Which one of the heads in the pair forms an AM complex is ambiguous at this stage

Even though for the reasons stated above, and also from time-resolved x-ray diffraction data (where the response of the interference spacing of the 3M has been followed during a quick release; unpublished data), we tend to favor the hypothesis that the more perpendicular head forms an AM complex, whereas the other one is ready to take over if/ when needed. Therefore it is the population of the former type of head that is responsible for the appearance of the actin-based layer lines during isometric contraction, whereas the latter type is responsible for the remnants of myosin layer lines at Po (Bordas et aL, 1993, 1999). However, at this stage one must leave open the possibility that the actual situation may be the other way around. If indeed that was the case, we note that the inclined and now assumed attached head (Fig. 6) is reminiscent of that in rigor (Whittaker et al., 1995). The other, more perpendicularly aligned, head exhibits, on the chosen view for the projection, the ~120 deg bent configuration of the nucleotide-free myosin head (Rayment et al., 1993). However, it is conceivable that an equally good fit to the mass projection might be obtained by bending the rigor head, via a hinged movement of its tail, such as it has been proposed from analysis of crystals of myosin fragments (Holmes 1997). If this were the case, we note that the swing of the tail between the two configurations shown in Fig. 6 would be close to 10.0 nm rather than the much smaller swing of ~3.5 nm deduced from electron microscopy (Whittaker et al., 1995).

Be what it may, the point at this stage is that to interpret x-ray diffraction data from muscle tissues, during the various mechanical protocols to which they are submitted, it is important to include in the modeling that the two myosin heads may have distinctly different axial orientation at the plateau of isometric contraction.

[Reference]

REFERENCES

[Reference]

Bassford, M. L. 2001. Modelling the molecular structure of muscle with comparison to x-ray diffraction data. Ph.D. thesis, University of Leicester, Leicester, UK. 190 pp.

Boesecke, P., O. Diat, and B. Rasmussen. 1995. High-brilliance beamline at the European Synchrotron Radiation Facility. Rev. Sci. Instrum 66:1636-1638.

Bordas, J., G. P. Diakun, F. G. Diaz-Banos, J. E. Harries, R. A. Lewis, J. Lowy, G. R. Mant, M. L. Martin-Fernandez, and E. Towns-Andrews. 1993. Two-dimensional time-resolved x-ray diffraction studies of live isometrically contracting frog sartorius muscle. J. Muscle Res. Cell Motif. 14:311-324.

Bordas, J., J. Lowy, A. Svensson, J. E. Harries, G. P. Diakun, J. Gandy, C. Miles, G. R. Mant, and E. Towns-Andrews. 1995. X-ray evidence that in contracting muscle there exist two distinct populations of myosin heads. Biophys. J. 68:99s-lOSs.

[Reference]

Bordas, J., A. Svensson, M. Rothery, J. Lowy, G. P. Diakun, and P. Boesecke. 1999. Extensibility and symmetry of actin filaments in contracting muscles. Biophys. J. 77:3197-3207.

Diaz-Banos, F. G., J. Bordas, J. Lowy, and A. Svensson. 1996. Small segmental rearrangements in the myosin head can explain force generation in muscle. Biophys. J. 71:576-589.

Dobbie, I., M. Linari, G. Piazzesi, M. Reconditi, N. Koubassova, M. A. Ferenczi, V. Lombardi, and M. Irving. 1998. Elastic bending and active tilting of myosin heads during muscle contraction. Nature. 396:383-387.

Ford, L. E., A. F. Huxley, and R. M. Simmons. 1981. The relation between stiffness and filament overlap in stimulated frog muscle fibres. J. Physiol. 311:219-249.

Haselgrove, J. C. 1975. X-ray evidence for conformational changes in the myosin filaments of vertebrate striated muscle. J. Mol. Biol. 92:113-143.

Holmes, K. C. 1997. The swinging lever-arm hypothesis of muscle contraction. Curr. BioL 7:8112-81 18.

[Reference]

Huxley, H. E. 1969. The mechanism of muscular contraction. Science. 164:1356-1366.

Huxley, A. F. 1974. Muscular contraction. J. PhysioL 243:1-43.

Huxley, H. E., and W. Brown. 1967. The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. MoL BioL 30:383-434.

Huxley, H. E., R. M. Simmons, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch. 1981. Millisecond time resolved changes in x-ray reflections from contracting muscle during rapid mechanical transients,

[Reference]

recorded using synchrotron radiation. Proc. Natl. Acad. Sci. U.S.A. 78:2297-2301.

Huxley, H. E., R. M. Simmons, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch. 1982. Time resolved x-ray diffraction studies of the myosin layer line reflections during muscle contraction. J. Mol. Biol. 158:637-684.

Huxley, H. E., R. M. Simmons, A. R. Faruqi, M. Kress, J. Bordas, and M. H. J. Koch. 1983. Changes in the x-ray reflections from contracting muscle during rapid mechanical transients and their structural implications. J. Mol. Biol. 169:469-506.

Huxley, H. E., A. Stewart, H. Sosa, and T. Irving. 1994. X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle. Biophys. J. 67:2411-2421.

Huxley, A. F., and S. Tideswell. 1996. Filament compliance and tension transients in muscle. J. Muscle Res. Cell Motil. 17:507-511.

Huxley, A. F., and S. Tideswell. 1997. Rapid regeneration of the power stroke in contracting muscle by attachment of second myosin head. J. Muscle Res. Cell Motil. 18:111-114.

Irving, M., V. Lombardi, G. Piazzesi, and M. A. Ferenczi. 1992. Myosin head movements are synchronous with the elementary force-generating process in muscle. Nature. 357:156-158.

[Reference]

Lombardi, V., G. Piazzesi, and M. Linari. 1992. Rapid regeneration of the actin-myosin power in contracting muscle. Nature. 355:638-641.

Malinchik, S. B., and V. V. Lednev. 1992. Interpretation of the x-ray diffraction pattern from relaxed skeletal muscle and modelling of the thick filament structure. J. Muscle Res. Cell Mot. 13:406-419.

Martin Fernandez, M. L., J. Bordas, G. P. Diakun, J. E. Harries, J. Lowy, G. R. Mant, A. Svensson, and E. Towns-Andrews. 1994. Time resolved x-ray diffraction studies of myosin head movements in live frog sartorius muscle during isometric and isotonic contractions. J. Muscle Res. Cell Motil. 15:319-348.

Rayment, I., W. R. Rypniewski, K. Schmidt-Base, R. Smith, D. R. Tomchick, M. M. Benning, D. A. Winklemann, G. Wesenberg, and H. M. Holden. 1993. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 261:50-58.

Rome, E. 1972. Structural studies by x-ray diffraction of striated muscle permeated with certain ions and proteins. Cold Spring Harbor Symp. Quant. Biol. 37:331-339.

Rome, E., T. Hirabayashi, and S. V. Perry. 1973a. X-ray diffraction of muscle labelled with antibody to troponin-C. Nat. New Biol. 244: 154-155.

[Reference]

Rome, E., G. Offer, and F. A. Pepe. 1973b. X-ray diffraction of muscle labelled with antibody to C-protein. Nat. New BioL 244:152-154.

Squire, J. M. 1981. The Structural Basis of Muscular Contraction. Plenum Press, New York.

Squire, J. M., J. J. Harford, A. C. Edman, and M. Sjostrom. 1982. Fine structure of the A-band in cryo-section: cross bridge distribution and the axial structure of the human C-zone. J. Mol. Biol. 155:467-494.

Vainshtein, B. K. 1966. Diffraction of X-rays by Chain Molecules. Elsevier, Amsterdam.

Wakabayashi, K., Y. Sugimoto, H. Tanaka, Y. Ueno, Y. Takesawa, and Y. Amemiya. 1994. X-ray evidence for the extensibility of actin and myosin filaments during muscle contraction. Biophys. J. 67:2422-2435.

Whittaker, M., E. M. Wilson-Kubalek, J. E. Smith, L. Faust, R. A. Milligan, and H. L. Sweeney. 1995. A 35-A movement of smooth muscle myosin on ADP release. Nature. 378:748-751.

[Author Affiliation]

J. Juanhuix,* J. Bordas,* J. Campmany,* A. Svensson,^ M. L. Bassford,^ and T. Narayanan^^

*Laboratori Llum Sincrotro-Institut Fisica Aites Energies, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain; ^Department of Physics and Astronomy, University of Leicester, Leicester LE1 7RH, United Kingdom; and ^^European Synchrotron Radiation Facility, F-38043 Grenoble, France

[Author Affiliation]

Received for publication 28 January 2000 and in final form 12 December 2000.

Address reprint requests to Dr. Joan Bordas, Edifici Ciencies Nord, LSBIFAE, Campus UAB, E-08193 Bellaterra, Spain. Tel.: 34-93-581-28-54; Fax: 34-93-581-32-13; E-mail: jbordas@ifae.es.

(C) 2001 by the Biophysical Society