Biomedical subjects

Go Ueno

Publications and source records attributed to Go Ueno.

5 recordsLinked to original sources

PubMed2006

Dose dependence of radiation damage for protein crystals studied at various X-ray energies.

Radiation damage to protein crystals is the most serious problem in obtaining accurate structures from protein crystallography. In order to examine the photon energy dependence of radiation damage, 12 to 15 data sets from each of nine tetragonal lysozyme crystals were collected at nine different X-ray energies (6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV) using beamline BL41XU at SPring-8. All results were compared on the basis of absorbed dose, expressed in Gray (Gy). Crystallographic statistics, such as the values of lattice constants, R(merge) and I/sigma(I), for each data set degraded at all nine energies as the exposure time for each crystal increased. In all data sets, radiation damage was observed after the absorbed dose exceeded 10(6) Gy. However, from the point of view of crystallographic statistics normalized to the absorbed dose, no clear dependence on photon energy was observed in these results. Structural refinement showed that the average B-factor for the last data set was larger than that for the first data set at all energies tested. However, no energy dependence of radiation damage on B-factor was found. Furthermore, disruption of disulfide bonds due to radiation damage was observed in electron density maps even at the highest photon energy (33 keV) used in this study. Therefore, these results suggest that radiation damage in the energy range investigated could be evaluated based on absorbed dose without energy dependence, and that it is important to minimize the absorbed dose in a crystal sample for obtaining an accurate protein structure.

Computer Simulation↗

PubMed2006

RIKEN structural genomics beamlines at the SPring-8; high throughput protein crystallography with automated beamline operation.

RIKEN Structural Genomics Beamlines, BL26B1 & BL26B2 at the SPring-8, have been constructed for the structural genomics research. The main feature of the beamline is full automation of the successive data collections to maximize the beam-time efficiency. The beamline optics adopted a standard design commonly used for the SPring-8 bending magnet beamlines. Beamline instruments are operated by centralized control system through the computer network to achieve the automatic operation. The core part of the beamline development is a sample management system composed of sample changer robots for laboratory and beamline, and a networked sample database. BL26B1 has started user operation, and the automatic operation with the sample management system has been implemented at BL26B2.

Crystallography, X-Ray↗

PubMed2005

Beamline Scheduling Software: administration software for automatic operation of the RIKEN structural genomics beamlines at SPring-8.

Beamline Scheduling Software (BSS) has been developed for the schedule management and equipment control of the RIKEN Structural Genomics Beamlines I and II (BL26B1 and BL26B2 at SPring-8). The beamline operation is automated with a sample-changer robot and a database system. The experimental schedule is registered to BSS as a list of diffraction measurements for numbers of protein crystals. BSS implements the beamline operation communicating with beamline instruments through the computer network. The RIKEN Structural Genomics Beamline I (BL26B1) opened for users using BSS in October 2002, and the automatic operation was implemented at BL26B2 in October 2003.

Automation↗

PubMed2005

A norovirus protease structure provides insights into active and substrate binding site integrity.

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.

3C Viral Proteases↗

PubMed2004

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop.

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.

15-Oxoprostaglandin 13-Reductase↗