Katsuaki Inoue

Small-angle X-ray scattering station at the SPring-8 RIKEN beamline

RIKEN beamline I (BL45XU) is an undulator beamline with two branches. One is for protein crystallography (PX) and the other is for small-angle x-ray scattering (SAXS). The beam is split into the two branches by a diamond monochromator so that two experiments can be done simultaneously [Yamamoto et al. (1995) Rev. Sci. Instrum. 66, 1833-1835]. The SAXS branch was designed for studying the weak interaction of proteins or subunits of fibrous or protein solutions especially using hydrostatic pressure. The optics makes use of the good parallelism of the undulator beam in order to reduce parasitic scattering. The beamline consists of a double crystal monochromator and a K-B type focusing mirror system. In order to cope with the high flux of the beam, an x-ray image intensifier (Hamamatsu Photonics, V5445P) with a cooled CCD camera (C4880-82) was used. As a result, decreases in both collection time and sample amount were realized in standard static experiments. These improvements will greatly facilitate SAXS experiments under high pressure.

Collapse of the hydration shell of a protein prior to thermal unfolding

Based on high statistical quality wide-angle X-ray scattering data for the unfolding–refolding process of hen egg-white lysozyme (HEWL), we have analysed the change of the hydration shell as a function temperature using the program CRYSOL. The present results suggest that the decrease of the hydration-shell density starts from a lower temperature than the transition temperature of the collapse of the tertiary structure of HEWL. Although the use of CRYSOL for scattering data for proteins before the transition has an apparent limitation, the collapse of the hydration shell prior to the unfolding of HEWL agrees with a slight tendency of the radius of gyration to decrease during the thermal unfolding process.

Structural hierarchy of several proteins observed by wide-angle solution scattering

In the present study using a high-intensity X-ray beam from a third-generation synchrotron radiation source, it is demonstrated that a wide-angle X-ray scattering (WAXS) profile from several globular proteins in solution can reflect not only the overall structures (approximately 300 A distance resolution) but also intramolecular structures ranging to secondary structures (approximately 2.5 A distance resolution). The proteins treated in the present experiments are classified as different types of structure categories, namely, as all-alpha, all-beta and alpha + beta proteins. Here the full-range experimental scattering curves are compared with the theoretical curves, suggesting a further availability of the SR-WAXS method for studies of structure hierarchy and the function of proteins in solutions.

Characterization of two-dimensional ultra-small- angle X-ray scattering apparatus for application to rubber filled with spherical silica under elongation

Two-dimensional ultra-small-angle X-ray scattering (2D-USAXS) apparatus at SPring-8 has been characterized. 2D-USAXS is a promising tool to study the structural change of the hierachical aggregate structure of fillers such as carbon black and silica particles in rubber. The aggregate structure of fillers is key to understanding the reinforcement effects which fillers show in rubber. We have applied 2D-USAXS to rubber filled with spherical silica particles and proved it to be a powerful technique.

Structural analysis of lipocalin-type prostaglandin D synthase complexed with biliverdin by small-angle X-ray scattering and multi-dimensional NMR.

Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD(2) synthase and an extracellular transporter for small lipophilic molecules. From a series of biochemical studies, it has been found that L-PGDS has an ability to bind a variety of lipophilic ligands such as biliverdin, bilirubin and retinoids in vitro. Therefore, we considered that it is necessary to clarify the molecular structure of L-PGDS upon binding ligand in order to understand the physiological relevance of L-PGDS as a transporter protein. We investigated a molecular structure of L-PGDS/biliverdin complex by small-angle X-ray scattering (SAXS) and multi-dimensional NMR measurements, and characterized the binding mechanism in detail. SAXS measurements revealed that L-PGDS has a globular shape and becomes compact by 1.3A in radius of gyration on binding biliverdin. NMR experiments revealed that L-PGDS possessed an eight-stranded antiparallel beta-barrel forming a central cavity. Upon the titration with biliverdin, some cross-peaks for residues surrounding the cavity and EF-loop and H2-helix above the beta-barrel shifted, and the intensity of other cross-peaks decreased with signal broadenings in (1)H-(15)N heteronuclear single quantum coherence spectra. These results demonstrate that L-PGDS holds biliverdin within the beta-barrel, and the conformation of the loop regions above the beta-barrel changes upon binding biliverdin. Through such a conformational change, the whole molecule of L-PGDS becomes compact.

Fast x-ray recordings reveal dynamic action of contractile and regulatory proteins in stretch-activated insect flight muscle.

To assess the ability of the thin-filament regulatory system to control each stretch-activation (SA) event in the fast beating of asynchronous insect flight muscle (IFM), we obtained fast (3.4 ms/frame) and semistatic (> or = 50 ms) x-ray diffraction recordings for IFM fibers from bumblebees (beating at 170 Hz) and compared the results with those acquired in giant waterbugs (20-30 Hz) and crane flies (40 Hz, semistatic only). In contrast to the well-documented large SA force of waterbug IFMs, the SA force of bumblebee and crane fly IFMs was small compared to their large isometric force. In semistatic recordings, step-stretched bumblebee and crane fly IFMs showed smaller net SA-associated intensity changes in reflections that report myosin attachment to actin and tropomyosin movement toward its activating position. However, fast recordings on bumblebee IFMs showed a fast and large temporary reversal of intensities in these reflections, suggesting that the myosin heads supporting isometric force are dynamically replaced by SA-supporting heads, and that tropomyosin moves to and back from its inactivating position in milliseconds. In waterbug IFMs, the fast temporary reversal of intensities was not obvious. The observed rates of the attachment/detachment of myosin heads and the motion of tropomyosin are fast enough for the thin-filament regulatory system to control each SA event in fast-beating insects.

Compact Packing of Lipocalin-type Prostaglandin D Synthase Induced by Binding of Lipophilic Ligands*

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi-functioning protein belonging to the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. In the present study, to clarify the conformational changes of lipocalin proteins induced by binding of lipophilic ligands, such as all-trans-retinoic acid (RA), bilirubin (BR) and biliverdin (BV), we measured small-angle X-ray scattering (SAXS) of L-PGDS and that of two other lipocalins, beta-lactoglobulin (betaLG) and retinol-binding protein (RBP). L-PGDS bound all three ligands with high affinity, while betaLG and RBP could bind only RA. The radius of gyration was estimated to be 19.4 A for L-PGDS, and 18.8 A for L-PGDS/RA, 17.3 A for L-PGDS/BR and 17.8 A for L-PGDS/BV complexes, indicating that L-PGDS became compact after binding of these ligands. Alternatively, the radius of gyration of betaLG and RBP was 20.3 and 26.2 A, respectively, and was almost the same before and after RA binding. Based on the SAXS data, we found that the compact packing upon binding ligands is a special feature of L-PGDS and it may be ascribed to the conformational flexibility of L-PGDS molecule itself, which underlies the high-affinity for its ligands.

SAXS, SANS and NSE studies on unbound state in DPPC/water/CaCl2 system

The temperature and CaCl 2 concentration dependence of the lamellar repeat distance of a dipalmitoylphosphatidylcholine (DPPC) aqueous solution was investigated by small-angle X-ray and neutron scattering, and neutron spin echo. At certain CaCl 2 concentrations, the repeat distance in the liquid-crystalline phase tends to increase up to infinity while that in the gel phase has an upper limit. This behavior is attributable to the fact that the steric repulsion of lipid bilayers due to the membrane undulation in the liquid-crystalline phase is larger than that in the gel phase. The free-energy calculation suggested that the cooperation of the short-range electrostatic interaction and the long-range steric interaction is the origin of the increase in the repeat distance in the liquid-crystalline phase, and it can be the origin of an “unbinding transition”.

Present Status of BL40B2 and BL40XU at SPring‐8 (Beamlines for Small Angle X‐ray Scattering)

BL40B2 and BL40XU were built at SPring‐8 in 1999. They are now open for public users and many kinds of small angle x‐ray scattering experiments are carried out using these beamlines. We report the present status of these two beamlines. Also some examples of experimental results that were obtained at these beamlines are shown. BL40B2 is for recording small‐angle x‐ray scattering from non‐crystalline biological materials. The light source is a bending magnet and the white x‐rays generated by are monochromatized using a double crystal monochromator and focused by a 1‐m‐long rhodium‐coated bent‐cylinder mirror. The photon flux is 1×1011 photons/sec at 12.4 keV. The tunable energy range is 7 keV ∼ 18 keV. As a detector, an imaging plate area detector (RIGAKU R‐AXIS IV++) is installed. Two fixed‐length vacuum paths allow camera lengths of 400 mm and 1000 mm. This beamline is suitable for static measurements with high accuracy and high resolution. The aim of BL40XU is to provide very high flux of x‐rays for vari...

Evolution of long-range myofibrillar crystallinity in insect flight muscle as examined by X-ray cryomicrodiffraction

Insect flight muscle is known for its crystal-quality regularity of contractile protein arrangement within a sarcomere. We have previously shown by X-ray microdiffraction that the crystal-quality regularity in bumble-bee flight muscle is not confined within a sarcomere, but extends over the entire length of a myofibril (>1000 sarcomeres connected in series). Because of this, the whole myofibril may be regarded as a millimetre-long, natural single protein crystal. Using bright X-ray beams from a synchrotron radiation source, we examined how this long-range crystallinity has evolved among winged insects. We analysed >4600 microdiffraction patterns of quick-frozen myofibrils from 50 insect species, covering all the major winged insect orders. The results show that the occurrence of such long-range crystallinity largely coincides with insect orders with asynchronous muscle operation. However, a few of the more skilled fliers among lower-order insects apparently have developed various degrees of structural regularity, suggesting that the demand for skilful flight has driven the lattice structure towards increased regularity.