CRYO-DIFFRACTION OF INSECT FLIGHT MUSCLE

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our program combines & compares X-ray diffraction (glycerinated native fibers) and thin-section EM tomography (fibers quick-frozen & freeze-substituted) to characterize structure, arrangement and dynamic choreography of myosin crossbridges (motor molecules of muscle) in a waterbug insect flight muscle (IFM) of unexcelled crystalline regularity. Revealing X-ray results will be checked by parallel EM of fibers quick-frozen during the same state or maneuver, to directly image the structural changes. (IFM gives the clearest EM images of myosin crossbridges of any muscle.) X-ray patterns from IFM differ in important ways from those of other striated muscles (frog skeletal) studied by XRD, owing to differences in filament lattice packing, in thick filament helical geometry, and a full-overlap sarcomere scheme limited to ~3% length changes. X-ray reflections (to ~5 nm) attributable to myosin filaments, actin filaments, and both jointly, are as well worked out as in frog muscle, but not yet so well studied in dynamic (contraction) states. Thanks to the beam quality, intensity and detector systems at the APS, much of the catch-up with frog muscle studies can be done in 2-4 years. The present proposal is to develop cryodiffraction of freeze-trapped dynamic transitions during contractile states activated by stretch or calcium to characterize myosin crossbridge actions that underlie force generation, force bearing, active shortening and braking resistance to forced lengthening. The purpose of this GUP proposal is to evaluate freezing conditions that can enhance sample lifetime so that diffraction patterns of highest resolution, showing even weakest reflections, can be obtained from small specimens before radiation damage sets in. Because of the weakness of the diffraction from the muscle system this study must be done on a synchrotrron source. As far as we know this has not been successfully achieved in muscle before and may be expected to lead to novel findings.