Mikio Kataoka

Aggregation of bovine serum albumin upon cleavage of its disulfide bonds, studied by the time-resolved small-angle x-ray scattering technique with synchrotron radiation

A rapid mixing system of the stopped-flow type, used with small-angle X-ray scattering equipment using synchrotron radiation, is described. The process of aggregation of bovine serum albumin was traced with a time interval of 50 s, initiated upon cleavage of its disulfide bonds by reduction with dithiothreitol. The results indicate that a 218-fold molar excess of dithiothreitol over the number of moles of disulfide bonds in bovine serum albumin is sufficient to initiate the reaction immediately after mixing, which reaches equilibrium in about 15 min. On the other hand, half this amount is not sufficient to initiate the reaction, so that the reaction is delayed by about 150 s. Such a single-shot time-resolved experiment showed that experiments with a time interval of 100 ms are possible with repeated multi-shot runs.

Crystallographic characterization by X-ray diffraction of the M-intermediate from the photo-cycle of bacteriorhodopsin at room temperature

The structure of the M‐intermediate appearing in the photo‐cycle of bacteriorhodopsin was studied with X‐ray diffraction techniques at room temperature. The lifetime of the M‐intermediate was prolonged by treatment with an arginine solution at alkaline pH (Nakasako et al.,FEBS Lett. 254, 211–214). The diffraction profile of membranes which had accumulated the M‐intermediate had small but significant differences in the intensities of Bragg reflections and the lattice constant in comparison with that of membranes havingtrans‐bacteriorhodopsin. Diffraction intensities were carefully evaluated and the structural changes during the formation of the intermediate were evaluated with difference Fourier analysis. We could find structural changes around helices G and B.

A model for the dimeric molecular structure of phytochrome based on small‐angle X‐ray scattering

The dimeric molecular structure of pea phytochrome (monomer molecular mass 114 kDa) in the red‐light‐absorbing form was studied with reference to the structures of its molecular domains in a monomer (an N‐terminal 59‐kDa chromophoric domain and a C‐terminal 55‐kDa nonchromophoric domain) using small‐angle X‐ray scattering, and a ‘four‐leaved shape’ model is proposed. The propriety of the model was confirmed by images of the molecule obtained by rotary shadowing electron microscopy.

Quaternary structure of pea phytochrome I dimer studied with small-angle X-ray scattering and rotary-shadowing electron microscopy

Abstract— The quaternary structure of pea phytochrome type I (PI) dimer in the red‐light‐absorbing form was studied by small‐angle X‐ray scattering (SAXS) technique and rotary‐shadowing electron microscopy. Structural parameters for PI 114 kDa chromopeptide dimer and its tryptically digested N‐terminal 59 kDa chromopeptide monomer, such as average electron density, molecular volume and the second moment of electron density distribution, were determined in terms of SAXS using the contrast variation method. Furthermore, by means of model simulation for the scattering profiles of the chromopeptides, most plausible structural models for both peptides were constructed. The distance between the chromophoric domains was estimated to be about 70 A in the resultant model for 114 kDa chromopeptide dimer. Furthermore, the model was consistent with the electron‐micrographic images of both the intact PI dimer and the PI 114 kDa chromopeptide dimer, so that the N‐terminal 7 kDa fragment did not significantly contribute the low‐resolution images of the dimer.

BACTERIORHODOPSIN RECONSTITUTED FROM TWO INDIVIDUAL HELICES AND THE COMPLEMENTARY FIVE-HELIX FRAGMENT IS PHOTOACTIVE

Abstract— Bacteriorhodopsin (bR), a light‐driven proton pump, consists of a bundle of seven membrane‐spanning alpha‐helices connected to each other by short extramembranous loops. Previously it has been shown that bR can be reconstituted from three fragments corresponding to the first helix, the second helix, and the remaining five helices, and that this reconstituted material reforms the native structure of bR. In this study, it is shown that the native function is also recovered. Low‐temperature spectroscopy was used to examine the photochemical properties of bR reconstituted from three fragments. At room temperature at pH 6, the reconstituted material shows essentially the same absorption spectrum as native bR, while upon raising the pH at room temperature or cooling the sample in glycerol, a second, blue‐shifted peak appears. The pH and temperature dependence of the absorption spectrum indicates that the reconstituted bR is in an equilibrium between two pigments, which we call P560 and P480. Both pigments convert to their own K intermediates, which differ in absorption maxima, upon illumination with green light at —180°C. Each K intermediate can be reverted to its initial state by light. Similarly, both pigments convert to their own M intermediates upon irradiation with yellow light at ‐77°C. The M intermediate of both species can be reverted only to P560 by light. Both pigments are therefore photoactive. These unique photochemical properties of bR reconstituted from three fragments may be attributable to the lack of a covalent linkage in the loop connecting the A and B helices, and thus possibly to a change in the orientation of the B helix.

Small-angle X-ray scattering studies on the macromolecular structure of the red-light-absorbing form of 121 kDa pea phytochrome and its 114 kDa chromopeptide

The macromolecular structure of 121 kDa pea phytochrome and its 114 kDa chromopeptide was studied by small-angle X-ray scattering (SAXS) and steric exclusion column chromatography. The molecular mass of the 114 kDa chromopeptide in the red-light-absorbing form (Pr) was determined to be 228 kDa in 100 mM potassium phosphate and 1 mM EDTA (pH 7.8) by SAXS, indicating that the chromopeptide is a dimer. The radius of gyration (Rg) of the chromopeptide was determined to be 53.8 A by Guinier analysis of SAXS. The molecular mass of a sphere with an Rg of 53.8 A is calculated to be 1140 kDa, which is far greater than the observed molecular mass for the 114 kDa chromopeptide. The discrepancy comes from the deviation of the molecular shape from a sphere and/or the dense distribution of electrons in the marginal area of the molecule. The apparent molecular mass of the chromopeptide was determined to be 314–318 kDa from steric exclusion column chromatography, indicating a nonglobular molecular shape. Assuming the overall shape is an ellipsoid of revolution, a prolate one (a = 24, b = 115 A and an oblate one (a = 85, b = 9 A), where a and b are the equatorial diameter and the half-axial length, respectively, are calculated to have an Rg of 53.8 A and a molecular mass of 228 kDa. The Rg of the 121 kDa phytochrome was 54.0 A, suggesting that the terminal polypeptide(s), which is iacking in the 114 kDa chromopeptide, contributes only slightly to the molecular dimension. The apparent molecular mass of the chromopeptide increased to 321–324 kDa after red-light irradiation, indicating that the far-red-light-absorbing form (Pfr) of the chromopeptide has a different molecular dimension from that of Pr. The Guinier analysis of the SAXS of the chromopeptide in Pfr, however, was not successful, owing to the low content of Pfr and the aggregation after saturating red-light irradiation.

Arginine remarkably prolongs the lifetime of the M‐intermediate in the bacteriorhodopsin photocycle at room temperature

The lifetime of the M‐intermediate in the bacteriorhodopsin photocycle was remarkably prolonged on drying a suspension of purple membrane, in arginine solution at alkaline pH. The M‐intermediate could be completely accumulated under illumination (530 nm) at room temperature. The crystalline structure of purple membranes was retained after this treatment. The lifetime of the M‐intermediate was found to be longer than 100 s and depended on the pH of the purple membrane suspension before drying. It was suggested that an interaction between the guanidium group of arginine and an amino acid residue played an important role in the prolongation.

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering.

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.

Instrumentation for the photoacoustic EXAFS method

The instrumentation for the photoacoustic EXAFS experiment detailed here is under development at the Photon Factory. In the experimental setup, simultaneous detection of the absorption spectra in addition to the photoacoustic measurement was useful for confirmation of this phenomena and for data correction. The current detection limit of this system is not sufficient for application to a wide variety of samples. Several ways for improvement of the quality of the spectra were carried out and the results were discussed.

Photoacoustic EXAFS of Solid Phase

Fine structures observed in X-ray photoacoustic spectra of various solids were compared with those in the absorption spectra. For a precise comparison, the photoacoustic spectra were corrected for energy-depending X-ray absorption of gas existing in the optical path. Finally corrected photoacoustic spectra showed a leveled or decreasing trend with increasing photon energy. Good correspondence between the X-ray photoacoustic spectra and the absorption spectra further confirmed that the fine structures in the photoacoustic spectra are identical with the extended X-ray absorption fine structures (EXAFS).