Small-Angle X-ray Diffraction of Muscle Using Undulator Radiation from the Tristan Main Ring at KEK

Abstract

Time-resolved X-ray diffraction of muscle has demanded ever-increasing flux into small sample volumes with low beam divergence. Results are reported of static and time-resolved small-angle X-ray diffraction studies on muscle fibers using a hard X-ray undulator installed in the Tristan main ring at KEK, Tsukuba, Japan, as an innovative source of synchrotron radiation more intense and better collimated than that available with the Photon Factory bending-magnet beamline. Static studies used the low divergence of the source to obtain detailed high-quality diffraction patterns of stable muscle states. The diffraction patterns from live skeletal muscles showed the numerous (over 100) meridional reflections. The well collimated beam from the undulator made it possible to clearly resolve, with an angular resolution of ca 700 nm, the closely spaced diffraction peaks arising from the two halves of the thick filaments centred on the M lines in a sarcomere, in addition, the diffraction peaks from the thin filaments on opposite sides of the Z bands could be resolved with an angular resolution of ca 1000 nm. The detailed structure of the meridional pattern defines the nature of the molecular packing in the thick and thin filaments. Time-resolved experiments using a focusing mirror aimed to prove cross-bridge states in striated muscle fibers by collecting X-ray diffraction data at a 0.185 ms time resolution from sinusoidally oscillating chemically skinned rabbit muscle fibers during active contraction and in rigor. When sinusoidal length changes at 500 Hz with a peak-to-peak amplitude of 0.6% of the muscle length were applied to a small fiber bundle, the tension showed a simple elastic response during the length oscillation. In the active muscle the intensity of the 14.5 nm myosin-based meridional reflection changed out of phase with the tension change during the oscillating length change. In contrast, in the rigor muscle it occurred in phase with the tension change. The high time-resolved experiments provide an insight into the coupling between conformational changes and force generation of the actomyosin cross-bridges. These studies provide a preview of the expected gains for muscle studies from the more widespread use of undulator radiation at third-generation synchrotron sources.

Backward Movements of Cross-Bridges by Application of Stretch and by Binding of MgADP to Skeletal Muscle Fibers in the Rigor State as Studied by X-Ray Diffraction

“…Recently, Burghardt et al (1997) also found that fluorescent probe bound to a Cys707 did not rotate when rigor muscle fibers were rapidly stretched. However, Tanaka et al (1991) and recently Yagi et al (1996) reported by x-ray diffraction that the 14.4-nm meridional intensity changed in response to tension changes when slow and fast sinusoidal length changes were applied to rigor muscles, respectively. Recently, Irving et al (1995) observed a small but significant change in the orientation of fluorescent dyes attached to the regulatory light chain in a myosin when the rigor muscle fiber was rapidly stretched, suggesting that the cross-bridge changes are confined to that region.…”

“…The changes also include the local change around a Cys707 residue on the catalytic domain of a head (Fajer et al, 1990;Burghardt et al, 1997) as well as in an S2 part. Such movements may also explain the oscillatory intensity changes of the 14.4-nm reflection when slow (Tanaka et al, 1991) and fast (Yagi et al, 1996) sinusoidal length perturbations were applied to rigor muscles. The smallness in the observed change would be because of the mass involved in the movement is only a small fraction of the total cross-bridge mass.…”

“…The smallness in the observed change would be because the mass involved in the movement is a relatively small fraction of the total crossbridge mass. It is worth mentioning that the intensity changes of the 14.4-nm-based reflections in a rigor muscle induced by stretch were in the opposite direction to those during stretch applied to an actively contracting muscle (Tanaka et al, 1991;Yagi et al, 1996), whereas the intensity change of the 5.9-nm reflection was in the same direction as it was in an actively contracting muscle (Takezawa, Sugimoto, Kobayashi, and Wakabayashi, in preparation). The relaxed muscle produces little change in any reflection during the oscillatory length changes, indicating that the rigor linkages are needed to cause the intensity changes.…”

Fourth generation light sources

“…Reducing the energy at which a given ring is operated can be used to reduce the emittance, taking advantage of the quadratic dependence of emittance on electron energy, as has been done at PEP [10] and TRIS-TAN [11]. However damping time constants increase and instability threshold currents decrease as energy is reduced, limiting the effectiveness of this approach.…”

“…They would cost much more than the lower energy rings discussed above, even to reach an emittance of about 0.3 nm-rad, which is much larger than the diffraction limit for hard X-rays. Limited use has been made of undulators on the large circumference PEP [10] and TRISTAN [11] rings as third generation sources. However both are now being converted to BFactories.…”