Tatzuo Ueki

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.

Crystal structure of N-carbamyl-d-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases

BACKGROUND N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

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.

The RIKEN structural biology beamline II (BL44B2) at the SPring-8

A SPring-8 bending magnet beamline, the RIKEN structural biology beamline II (BL44B2), is dedicated to monochromatic/Laue macromolecular crystallography and X-ray absorption fine structure studies. The design and the performance of the beamline are presented. # 2001 Elsevier Science B.V. All rights reserved.

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild‐type RNase A and three mutants—F120A, F120G, and F120W—were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild‐type RNase A, except for the C‐terminal region of F120G with a high B‐factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild‐type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild‐type RNase A, and those of F120W and F120G appeared to be “inside out” compared with that of wild‐type RNase A. Only ∼1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

The optically active center and its activation process in Er-doped Si thin film produced by laser ablation

The local structure of erbium-doped silicon produced by the laser ablation technique is investigated by Er LIII-edge x-ray absorption fine structure analysis. The combined analysis of extended x-ray absorption fine structure analysis and an x-ray absorption near-edge structure simulation based on multiple-scattering theory reveals the most probable atomic coordination of the optically active center; Er bonded with six oxygen atoms has a C4v symmetry. The optical activation process of this system is also discussed. The Si target with 10 wt% Er2O3 has two kinds of local structures, C-rare-earth Er2O3 grain and another Er phase incorporated in Si. The laser ablation homogenizes these phases, and deposits a new single-phase structure of the octahedron (Oh point group) on the substrates. In this phase, the optical transition probability is low due to the forbidden 4f transition of Er in the crystal field originating from the higher-order symmetry of O. After annealing, degradation of the symmetry from Oh to C4...

Polyhedral assembly of a membrane protein in its three-dimensional crystal

A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.

Relationship between growth temperature of Anacystis nidulans and phase transition temperature of its thylakoid membranes

The temperatures of the lipid phase transition at which the solid phase disappears were determined by using the X-ray diffraction method in thylakoid membranes of the blue-green alga, Anacystis nidulans. The temperatures were determined as 26 and 16 degrees C for cells grown at 38 and 28 degrees C, respectively.

X‐ray optics research and development for SPring‐8 beamlines

X‐ray optics research and development for SPring‐8 beamlines are described. These include standard developments of monochromators, high‐heat load optics, wide energy‐range monochromators for bending beamlines, long x‐ray mirrors, and single and graded d‐spacing synthetic multilayers.

Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.