Katsuzo Wakabayashi

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.

Design of small-angle X-ray diffractometer using synchrotron radiation at the photon factory

Abstract A demagnifying focusing mirror-monochromator optics has been designed and constructed for time-resolved X-ray diffraction studies of biological substances. Focusing in the vertical plane is performed by a sequence of seven 20 cm long bent mirrors. Focusing in the horizontal plane is performed by a bent triangular silicon crystal monochromator which is placed downstream of the mirror. Phase space calculations predict that the optics should produce a focus 1.0 mm (V)×2.6 mm (H) (4σ-value) at the focus point. When the PF storage ring operates at 2.5 GeV and 0.5 A, the intensity is expected to be 10 11 –10 12 photons/s. A small-angle resolution of better than 1000 A should be obtainable at 1.5 A wavelength with an angular resolution of 0.8 mrad and 0.6 mrad in the vertical and horizontal directions, respectively.

Large‐aperture TV detector with a beryllium‐windowed image intensifier for x‐ray diffraction

A large‐aperture (150 mm and 230 mm in diameter) x‐ray TV‐type detector has been developed for x‐ray diffraction with synchrotron radiation. The detector consists of a beryllium‐windowed x‐ray image intensifier, an optical lens, a charge coupled device (CCD) image sensor, and data acquisition system. The spatial resolution is 270 μm(FWHM), and the dynamic range is 6000:1. The noise level is quantum limited. The nonuniformity of response and image distortion is corrected by software. When a TV‐rate (NTSC‐mode) CCD is used as an image sensor, time‐resolved measurements with a rate of 30 frame/s can be achieved with its noise quantum limited.

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study.

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.

Two-crystal structures of tropomyosin C-terminal fragment 176-273: exposure of the hydrophobic core to the solvent destabilizes the tropomyosin molecule.

Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, and when associated with troponin, it is responsible for the actin filament-based regulation of muscle contraction in vertebrate skeletal and cardiac muscles. It is widely believed that Tm adopts a flexible rod-like structure in which the flexibility must play a crucial role in its functions. To obtain more information about the flexibility of Tm, we solved and compared two crystal structures of the identical C-terminal segments, spanning approximately 40% of the entire length. We also compared these structures with our previously reported crystal structure of an almost identical Tm segment in a distinct crystal form. The parameters specifying the local coiled-coil geometry, such as the separation between two helices and the local helical pitch, undulate along the length of Tm in the same way as among the three crystal structures, indicating that these parameters are defined by the amino acid sequence. In the region of increased separation, around Glu-218 and Gln-263, the hydrophobic core is disrupted by three holes. Moreover, for the first time to our knowledge, for Tm, water molecules have been identified in these holes. In some structures, the B-factors are higher around the holes than in the rest of the molecule. The Tm coiled-coil must be destabilized and therefore may be flexible, not only in the alanine clusters but also in the regions of the broken core. A closer look at the local staggering between the two chains and the local bending revealed that the strain accumulates at the alanine cluster and may be relaxed in the broken core region. Moreover, the strain is distributed over a long range, even when a deformation like bending may occur at a limited number of spots. Thus, Tm should not be regarded as a train of short rigid rods connected by flexible linkers, but rather as a seamless rubber rod patched with relatively more flexible regions.

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.

X-ray structure analysis of the thin filament of crab striated muscle in the rigor state

X-ray diffraction studies were made on glycerinated leg striated muscles from the marine crab Portunus trituberculatus , in the rigor and relaxed states. In the rigor state, many distinct layer-lines with basic period 76·5 nm due to the thin filaments were observed, well-separated from several relatively weak layer-lines withbasic period 14·5 nm caused by the thick filaments. In the relaxed state, most of the layer-lines due to the thin filaments became very weak. No lattice sampling effect was observed on any layer-line, except on the equator. Layer-line spacings proved that the F-actin helix in the thin filament has 28 subunits in 13 turns. Structure analysis of the thin filament in the rigor state was done by using 13 layer-line reflections measured with reasonably good accuracy. The analysis was helped considerably by a difference Patterson function ΔQ ( r ) defined by ΔQ ( r )= Q ( r )−(1/ c ) λ 0 c Q ( r )dz where Q ( r ) is the Patterson function, r the position vector, z the co-ordinate parallel to the fibre axis and c the period of the filament. Results showed that, in the rigor state, the myosin heads attach periodically along the thin filament: they are bound to 3, 3′, 4, 4′ and 10, 10′, 11, 11′ actin monomers when the actin monomers are numbered 1 to 14 along one strand of the long-pitch helix and 1′ to 14′ along the other, 1′ being neighboured by 1 and 2. Troponin molecules sit on the filament with their centres of gravity at roughly the same z co-ordinates as 1, 1′ and 8, 8′ actin monomers, or as 5′, 6 and 12′, 13 as another possibility. Several theorems are given concrning ΔQ(r, z) , which may be generally useful in analysis of fibre structures.

Lateral forces in the filament lattice of vertebrate striated muscle in the rigor state.

The repulsive pressure between filaments in the lattice of skinned rabbit and frog striated muscle in rigor has been measured as a function of interfilament spacing, using the osmotic pressure generated by solutions of large, uncharged polymeric molecules (dextran and polyvinylpyrrolidone). The pressure/spacing measurements have been compared with theoretically derived curves for electrostatic pressure. In both muscles, the major part of the experimental curves (100-2,000 torr) lies in the same region as the electrostatic pressure curves, providing that a thick filament charge diameter of approximately 30 nm in rabbit and approximately 26 nm in frog is assumed. In chemically skinned or glycerol-extracted rabbit muscle the fit is good; in chemically skinned frog sartorius and semitendinosus muscle the fit is poor, particularly at lower pressures where a greater spacing is observed than expected on theoretical grounds. The charge diameter is much larger than the generally accepted value for thick filament backbone diameter. This may be because electron microscope results have underestimated the amount of filament shrinkage during sample preparation, or because most of the filament charge is located at some distance from the backbone surface, e.g., on HMM-S2. Decreasing the ionic strength of the external solution, changing the pH, and varying the sarcomere length all give pressure/spacing changes similar to those expected from electrostatic pressure calculations. We conclude that over most of the external pressure range studied, repulsive pressure in the lattice is predominantly electrostatic.

Time-resolved x-ray diffraction studies on the intensity changes of the 5.9 and 5.1 nm actin layer lines from frog skeletal muscle during an isometric tetanus using synchrotron radiation

Time-resolved x-ray diffraction studies have been made on the 5.9- and 5.1-nm actin layer lines from frog skeletal muscles during an isometric tetanus at 6 degrees C, using synchrotron radiation. The integrated intensities of these actin layer lines were found to increase during a tetanus by 30-50% for the 5.9-nm reflection and approximately 70% for the 5.1-nm reflection of the resting values. The intensity increase of both reflections was greater than that taking place in the transition from rest to rigor state. The intensity change of the 5.9-nm reflection preceded those of the myosin 42.9-nm off-meridional reflection and of the equatorial reflections, as well as the isometric tension development. The intensity profile of the 5.9-nm layer line during contraction was found to be different from that observed in the rigor state.

Stopped‐flow apparatus for x‐ray scattering at subzero temperature

A stopped‐flow apparatus was constructed for x‐ray scattering study at subzero temperature. It can be operated over a wide temperature range down to −20 °C with highly viscous solution (20 cP) successfully. We have applied the stopped‐flow x‐ray scattering method to many biological reactions. In particular, the association of E. coli ribosomal subunits was detected at −10 °C which was too fast to be detected at room temperature. Dissociation of E. foetida hemoglobin was measured by the stopped‐flow x‐ray scattering method combined with a time‐resolved imaging plate as a detector.