Yoji Inoko

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.

Structural Parameters of Dipalmitoyl Phosphatidylcholine Lamellar Phases and Bilayer Phase Transitions

Thicknesses d l of the lipid bilayer and d w of the water layer were determined separately with X-rays for dipalmitoyl phosphatidylcholine lamellar phase. In both cases of the presence and the absence of excess water, d l does not change appreciably at the pretransition (35°C) but drops down by about 5 A at the main transition (42°C) with increasing temperature. In the presence of excess water, d w jumps up by 4.0 A at the pretransition and by 2.0 A at the main transition. These results prove that there is practically no change of the tilting angle of hydrocarbon chains at the pretransition and that the remarkable increase of the repeat distance of the lamellar phase at the pretransition in the presence of excess water is caused by the increase of the thickness of the water layer.

The use of a Hamamatsu X-ray image intensifier with a cooled CCD as a solution X-ray scattering detector

CCD detectors are now widely used in many synchrotron small-angle X-ray scattering beamlines. The use of an X-ray image intensifier with cooled CCD (XR-II + CCD) was studied, especially for use in synchrotron solution X-ray scattering. Two samples, polystyrene latex and apoferritin, were used. These two samples have fine structure in the solution scattering profile due to symmetry and narrow size distribution. The recorded scattering profile, in comparison with that obtained by a position-sensitive proportional counter (PSPC), showed that XR-II + CCD has a much smaller practical dynamic range (100:1) than that of a pixel well (7500:1). This limited dynamic range was overcome by placing various-size masks on the detection plane, thereby eliminating the high-intensity region. The images recorded with various masks were combined, and the reconstituted solution scattering profile was submitted to various analyses, including Guinier analysis, power-law analysis, size distribution analysis and calculation of radial density distributions. The results were the same as those obtained with the PSPC. This indicates that spatial distortion as well as shading, a decrease in sensitivity from the centre to the edge of the detecting region [Amemiya, Ito, Yagi, Asano, Wakabayashi, Ueki & Endo (1995). Rev. Sci. Instrum. 66, 2290–2294], have very little effect on the SAXS results. This paper presents a practical protocol for obtaining a reliable solution scattering profile given the limitations of XR-II + CCD for synchrotron solution X-ray scattering.

Upgrade of the small angle X-ray scattering beamlines at the Photon Factory

BL-10C and BL-15A at the Photon Factory, which became operational in 1982, are some of the oldest small angle X-ray scattering beamlines in the world. Recently, both beamlines were upgraded for two-dimensional (2D) SAXS-WAXS experiments. A wide-area imaging plate (IP) detector and a fast-readout flat panel (FP) detector were installed at BL-10C and BL-15A, respectively. Preliminary experiments of both systems showed promising results.

Size-exclusion chromatography combined with small-angle X-ray scattering optics

Size-exclusion chromatography with on-line synchrotron radiation solution small-angle X-ray scattering optics, absorbance and/or refractive index detectors was evaluated by protein characterizations. The radius of gyration value and zero-angle scattering intensity of protein molecules eluted from the chromatography column were estimated using this measurement system. In addition, the characterization of the conformation of the eluted proteins was demonstrated for hen egg lysozyme and bovine submaxillary mucin. The present technique will be useful for not only the determination of the radius of gyration value and molecular weight of proteins with dimensions of 1-10 nm, but also for the structural characterization of the macromolecules during the chromatography.

How the formation process influences the structure of BChl c aggregates

The change of absorption spectra has been measured during the drying process of (31 R)bacteriochlorophyll (BChl) c from diethyl ether, dichloromethane (CH2Cl2) and carbon tetrachloride (CCl4) solutions. Absorption maxima of the Qy(0–0) transition of BChl c appear at 659 nm in diethyl ether, 680 nm in CH2Cl2 and 710 nm in CCl4. All these peaks are red-shifted to about 740 nm with formation of solid high aggregates when the solutions are completely dried. Fourier transform infrared spectra of the three solid aggregates are almost identical. However, magnetic circular dichroism and circular dichroism spectra are different and can be explained in terms of variations in stacking size of the aggregates and molecular arrangement of BChl c. Small-angle X-ray diffraction has been observed only for the aggregates treated with CH2Cl2, and the same sample gave rise to highly resolved cross polarization/magic angle spinning 13C nuclear magnetic resonance spectrum. The results suggest that molecular ordering of the solid-state BChl c aggregates is highly dependent on the formation process which is largely determined by the solvent used.

Small-angle light and X-ray scattering measurements of a protein–oligosaccharide complex mucin in solution

The molecular assembly and the chain conformation of intact bovine submaxillary mucin (BSM) in solution over wide-ranging concentrations were characterized by using low-angle laser light scattering and small-angle X-ray scattering (SAXS) methods. The specific refractive index increment of BSM was estimated to be 0.152 ml g−1 and was used to determine the molecular weight of BSM by low-angle laser light scattering photometry combined with high-performance gel chromatography. The total molecular weight of BSM was 55 million and the molecular weight of the main fractionated components was about 2 million. Fractal analysis of the SAXS data revealed that the intact BSM molecule is a chain with excluded volume (fractal dimension 1.67) at concentrations of 1.4 and 3.6 mg ml−1 and a chain with a Gaussian chain character (fractal dimension 2) at concentrations of 7.2–15 mg ml−1. Moreover, the Kratky plots of the SAXS data showed that the chain conformation of BSM molecules is a Gaussian (unfolded) structure in solution. The estimated cross-sectional radius of gyration value lay in the range 0.65–0.76 nm, which is reasonable for a long thin shape.

Small-Angle X-Ray Scattering Analysis of Membrane Protein Porin

The native (intact) and denatured (unfolded) states of a trimeric integral membrane protein Porin were characterized by synchrotron radiation small-angle X-ray scattering. Native trimeric Porin in the presence of SDS showed a scattering curve with two peaks at q=1.5 and 2.4 nm-1. The scattering curve of denatured monomeric Porin had a single peak at q=1.6 nm-1 and was similar to a typical scattering pattern for the water-soluble protein polypeptide in SDS. The radius of gyration value of denatured monomeric Porin in SDS was much the same as that of its native trimer (ca. 4 nm). The patterns of Kratky plots at the native and denatured state were different from each other. This finding will be useful for the kinetic study in synchrotron radiation small-angle X-ray scattering experiments to understand the folding and assembly mechanism of the integral membrane protein.

Characterization of a large glycoprotein proteoglycan by size-exclusion chromatography combined with light and X-ray scattering methods

The molecular weight and chain conformation of a proteoglycan derived from shark cartilage in solution were characterized by size-exclusion chromatography combined with low-angle laser light scattering and small-angle X-ray scattering methods. The total molecular weight of the proteoglycan was 3.9±0.2 million and the molecular weight of the main component was about 2.0±0.2 million. The X-ray scattering data revealed that the main components of the proteoglycan are nearly equal to a chain with excluded volume and their persistence lengths range from 13.5 to 16.4nm. These results show that size-exclusion chromatography combined with low-angle laser light scattering and small-angle X-ray scattering measurements are complementarily useful for characterization of large biopolymers in solution.

Soft X-Ray Small-Angle Scattering by Polystyrene Latexes Using Synchrotron Radiation

Small-angle scattering of soft X-rays by polystyrene latex spheres was studied using the synchrotron radiation from the electron synchrotron operated at about 1 GeV at the Institute for Nuclear Study, University of Tokyo. The exposure time required to make three smallest-angle diffraction peaks visible on a photographic plate was 2 min with the soft X-rays (46.8 A) of the synchrotron radiation, compared to 400 min with the CKα radiation (44.8 A) from the X-ray tube operated at 3 kV ×50 mA. The diffraction patterns were so distinct that inter-particle interference functions could be derived with four diffraction peaks. Experimental results as a whole suggest that soft X-rays in the synchrotron radiation will be very useful in studying two-dimensional structures of thin organic materials with the thickness of microns or submicrons.