ABSTRACT:CCP13 Workshop - Grenoble 2004

Low-angle X-ray Diffraction Studies of Muscle using the SRS, ESRF and APS

John M. Squire1, Carlo Knupp1,2, Manfred Roessle3, Hind A. AL-Khayat1, Michael K. Reedy4, Thomas C. Irving5, Felicity Eakins1 & Jeff Harford1,6 1Biological Structure & Function Section, Biomedical Sciences Division, Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ.
2School of Optometry and Vision Sciences, University of Cardiff, Cardiff CF10 3NB, Wales.
3ESRF, 6 rue Jules Horowitz, 38000 Grenoble, France.
4Dept of Cell Biology, Duke University, Durham, NC 27710, USA
5BioCAT, Dept. Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA
6Biological Sciences Department, Faculty of Life Sciences, Imperial College London, Exhibition Road, London SW7 2AZ.

The highly ordered but different striated muscles found in certain vertebrates, such as bony fish, and in some invertebrates, such as the insect asynchronous flight muscles in the giant water bug Lethocerus, provide enormously rich low-angle X-ray diffraction patterns which in some cases can be fully 'solved' to yield the dispositions of some of the component molecules in the muscle sarcomeres (Hudson et al., 1997; Squire et al., 1998, 2003(a-d); AL-Khayat et al., 2003). Analysis of the diffraction patterns from resting muscle has already shown that the myosin head shapes and distributions on the myosin filament surfaces in these two muscle types are different, but they both contrive to place the motor (catalytic) domains of some of the outermost myosin heads in a configuration that is almost what is needed for direct attachment of the heads to the adjacent actin filaments.New results from bony fish muscle using the very small and highly collimated X-ray beam on line ID-02 at the ESRF in Grenoble (Squire et al., 2004) reveal the presence of numerous hitherto unseen X-ray reflections.These promise to provide information not just about the myosin heads, but also about other components of the muscle A-band, namely C-protein (MyBP-C; Squire et al., 2003c) and titin (Connectin) on the myosin filaments, and troponin on the actin filaments.In addition, time-resolved X-ray diffraction studies can help to reveal the sequence of events that takes place on the onset of activation of an intact muscle and the nature of the structural changes that may be involved in force generation. The latest X-ray diffraction results on these two muscle systems will be reported and preliminary interpretations given.

AL-Khayat, H.A., Hudson, L., Reedy, M.K., Irving, T.C. & Squire, J.M. (2003) "Myosin head configuration in relaxed insect flight muscle: X-ray modelled resting crossbridges in a pre-powerstroke state are poised for actin binding." Biophys. J. 85, 1063-1079.
Hudson, L., Harford, J.J., Denny, R.J. & Squire, J.M. (1997) "Myosin head configurations in relaxed fish muscle: resting state myosin heads swing axially by 150Å or turn upside down to reach rigor". J. Mol. Biol. 273, 440-455.
Squire, J.M., Cantino, M., Chew, M., Denny, R., Harford, J.J., Hudson, L. & Luther, P.K.(1998) "Myosin rod packing schemes in vertebrate muscle thick filaments" J. Struct. Biol. 122, 128-138.
Squire, J.M., Knupp, C., AL-Khayat, H.A. & Harford, J.J. (2003a) "Millisecond time-resolved low-angle X-ray diffraction: a powerful, high-sensitivity technique for modelling real-time movements in biological macromolecular assemblies." Fibre Diffraction Review 11, 28-35.
Squire, J.M., AL-Khayat, H.A., Harford, J.J., Hudson, L., Irving, T.C., Knupp, C., Mok, N-S. & Reedy, M.K. (2003b) "Myosin filament structure and myosin crossbridge dynamics in fish and insect muscles."In 'Molecular and cellular aspects of muscle contraction' (Ed. H. Sugi).Kluwer/ Plenum (in press)
Squire, J.M., Luther, P.K. & Knupp, C. (2003c) "Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain."J. Mol. Biol. 331, 713-724.
Squire, J.M., AL-Khayat, H.A., Harford, J.J., Hudson, L., Irving, T., Knupp, C. & Reedy. M.K. (2003d) "Modelling muscle motor conformations using Low-angle X-ray diffraction". IEE Proc. BioNanotechnology (in press).
Squire, J.M., Roessle, M. & Knupp, C. (2004) "New X-ray diffraction observations on the structure of the vertebrate muscle A-band with possible interpretations based on C-protein (MyBP-C), titin, nebulin and troponin"(J. Mol. Biol. - in preparation).