A Gasless Reservoir Solution for Electro-Hydraulic Compact Drives With Two Prime Movers

Due to an increased focus on improving the energy efficiency and compactness of hydraulic linear actuators, the electro-hydraulic compact drive (ECD) has received increased attention lately. In this study the ECD consists of variable-speed electric motors and fixed-displacement pumps, which are directly connected to the cylinder, thus controlling the linear motion in a throttleless manner. Furthermore, ECDs are self-contained systems, i.e. based on a fully enclosed oil circuit, in order to avoid external contamination and air to enter the system and to increase system compactness. Conventionally a low-pressure gas-loaded accumulator is used as an oil reservoir to compensate for the flow imbalance occurring whenever utilizing single-rod cylinders in closed systems. The accumulator pressure is to be kept relatively low to stay within the required limits governed by the permitted pump housing’s pressure. Generally, this pressure is not allowed to exceed 1–3 bar. To avoid violating this limitation, the gas volume must be significantly larger than the actual oil volume, which needs to be stored in the accumulator. This requirement decreases the obtainable compactness of the ECD, especially for systems with a large cylinder stroke. Furthermore, the accumulator represents a potential of gas leakage, which ultimately could result in the ECD being non-functional. This paper presents a gasless reservoir solution, improving the system compactness and avoiding the risk of gas leakage. The proposed solution is based on a bootstrap reservoir which is charged by the lowest cylinder chamber pressure. This strategy is feasible for the class of ECDs that is capable of controlling the lowest cylinder chamber pressure alongside the cylinder motion. An ECD consisting of two electric prime movers is considered as a case study. It is shown how the gasless reservoir may be integrated into the system, and an analysis of how this affects the operating range and the dynamic couplings of the system is presented. This leads to the derivation of a control strategy for the Multi-Input-Multi-Output (MIMO) system based on state decoupling, by defining virtual inputs to control virtual outputs. A numerical study suggests that the reservoir volume may be reduced by approximately 50% for the given system dimensions. The proposed control strategy shows good position tracking performance while also being able to control the reservoir pressure within the pre-defined limits of 1 to 3 bar.

Pteridine glycosyltransferase from Chlorobium tepidum: crystallization and X-ray analysis

The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2′-yl)-β-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Å resolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c = 105.92 Å, β = 120.5°.

Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein fromStreptomyces fradiae

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation inStreptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced inEscherichia coliand purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space groupP3121, with unit-cell parametersa=b= 126.62,c= 95.63 Å.

Envelope protein VP24 fromWhite spot syndrome virus: expression, purification and crystallization

White spot syndrome virus(WSSV) is a major shrimp pathogen known to infect penaeid shrimp and other crustaceans. VP24 is one of the major envelope proteins of WSSV. In order to facilitate purification, crystallization and structure determination, the predicted N-terminal transmembrane region of approximately 26 amino acids was truncated from VP24 and several mutants were prepared to increase the proportion of selenomethionine (SeMet) residues for subsequent structural determination using the SAD method. Truncated VP24, its mutants and the corresponding SeMet-labelled proteins were purified, and the native and SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of VP24 were obtained using a reservoir consisting of 0.1 MTris–HCl pH 8.5, 2.75 Mammonium acetate with a drop volume ratio of two parts protein solution to one part reservoir solution. Notably, ATP was added as a critical additive to the drop with a final concentration of 10 mM. Crystals of SeMet-labelled VP24 mutant diffracted to 3.0 Å resolution and those of the native diffracted to 2.4 Å resolution; the crystals belonged to space groupI213, with unit-cell parametersa=b=c= 140 Å.

Crystallization and preliminary X-ray crystallographic analysis of Z-ring-associated protein (ZapD) fromEscherichia coli

Bacterial cytokinesis is accomplished by the Z-ring, which is a polymeric structure that includes the tubulin homologue FtsZ at the division site. ZapD, a Z-ring-associated protein, directly binds to FtsZ and stabilizes the polymerization of FtsZ to form a stable Z-ring during cytokinesis. Structural analysis of ZapD fromEscherichia coliwas performed to investigate the mechanism of ZapD-mediated FtsZ stabilization and polymerization. ZapD was crystallized using a reservoir solution consisting of 1.5 Mlithium sulfate, 0.1 MHEPES pH 7.8, 2%(v/v) polyethylene glycol 400. X-ray diffraction data were collected to 2.95 Å resolution. The crystals belonged to the hexagonal space groupP64, with unit-cell parametersa=b= 109.5,c= 106.7 Å, γ = 120.0°. Two monomers were present in the asymmetric unit, resulting in a crystal volume per protein mass (VM) of 3.25 Å3Da−1and a solvent content of 62.17%.

Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase fromDeinococcus radiodurans

In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress.Deinococcus radioduransis an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome ofDeinococcusshows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE fromD. radioduransare reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mMsodium acetate pH 5.0, 10% PEG 8000, 15–20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to beP21221, with unit-cell parametersa= 47.91,b= 62.94,c= 86.75 Å, α = β = γ = 90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%.

Crystal structures of bacterial flavin reductase GraD and its complex with NADH

Rhizobium sp. strain MTP-10005 uses the aromatic compound γ-resorcylate as a sole source of carbon and energy for growth. Resorcinol hydroxylase, which converts resorcinol to hydroxyquinol, plays an important role in the aerobic microbial catabolism of γ-resorcylate. Resorcinol hydroxylase from Rhizobium sp. strain MTP-10005 is a two-component enzyme consisting of the reductase and the monooxygenase components. The reductase component (GraD) is an oxidoreductase containing a flavin molecule as a cofactor. GraD catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the monooxygenase component GraA to hydroxylate resorcinol to hydroxyquinol. We have determined the three-dimensional structures of recombinant GraD with a bound FAD and in complex with NAD. GraD was crystallized at 293 K by the sitting-drop vapour-diffusion method using a precipitant solution containing 13 - 14% (w/v) PEG 2000, 6 - 9% (v/v) 2-propanol, 100 mM sodium citrate pH 5.6, 100 mM DTT and 200 μM FAD. The approximate dimensions of the obtained crystals were 0.1 × 0.1 × 0.15 mm3. The crystal diffracted to 1.8Å and belongs to space group P41212 with unit-cell parameters of a = b = 77.8 Å and c = 124.2 Å. The crystal structure has been determined by the molecular replacement and refined at 1.8 Å resolution. GraD exists as a homodimer, and each monomer contains an FAD. The probable binding site for NADH is covered with the N-terminal sub-domain in chain A, whereas the site is completely exposed to bulk solvent in chain B. The NAD-complex crystals were prepared by soaking the GraD crystals in the reservoir solution supplemented with NADH. The crystal diffracted to 1.8 Å, and the crystal structure was determined at 1.8 Å resolution. The Fo-Fc maps for the crystal soaked with NADH showed the electron densities corresponding to the nicotinamide ring and the adenyl moiety in chain B.

Crystal structure analyses of oxygenase component of resorcinol hydroxylase

The resorcinol hydroxylase is involved in the first step of the resorcinol catabolic pathway and catalyzes hydroxylation of resorcinol to hydroxyquinol. The enzyme belongs to the two-component flavin-diffusible monooxygenase family and acts in the coexistence of two components: an oxygenase and a flavin reductase. The oxygenase component hydroxylates the substrate using molecular oxygen and reduced flavin produced by the reductase. To understand the structural basis for the catalytic mechanism, we analyzed the crystal structure of the oxygenase component (GraA) from Rhizobium sp. strain MTP-10005. The GraA subunit has 409 amino acid residues. Apo-form crystals were obtained in the tetragonal space group I4122 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and K2HPO4. Holo-form crystals were obtained in the trigonal space group P3221 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and KNO3. Both structures were determined by molecular replacement and refined at 2.3 Å and 3.2 Å resolutions, respectively. GraA is a homotetramer with three molecular two-fold axes identical to crystallographic two-fold axes in the apo-form crystal. In the holo-form crystal, four tetramers exist in the asymmetric unit and each subunit binds one FAD. The subunit consists of three domains. The N-terminal domain has an α-structure mainly of antiparallel α-helices; the central domain has a β-structure of two β-sheets stacked together; the C-terminal domain has a four-helix-bundle structure of long antiparallel α-helices involved in tetramer formation. In the holo-form, the FAD is located in the space that is encompassed by these three domains. The loop region of 13 residues, which is disordered in the apo-form, is ordered and covers FAD of another subunit. The turn portion of the loop occludes the entrance of the putative active site.

Weak interaction of an inhibitor in the 20S proteasome

Proteasomes are the multicatalytic protein complexes with huge molecular weight. It is well known that the ubiquitin proteasome system plays an important role in regulated proteolysis. The 26S proteasome is composed of two 19S regulating components and a 20S proteasome. The 20S proteasome forms barrel shape and is composed of four rings, α- and β-rings; each ring contains highly homological seven α-subunits (1~7) and seven β-subunits (1~7), N-terminal tails of α-ring subunit form a gate to facilitate substrate entry. Each of 1, 2 and 5 β–subunit has different enzyme activity; 1 is caspase-, 2 tryptic- and 5 chymotryptic- like activity, respectively. We found a new protease inhibitor compound-A, which interacts weakly against proteasome. In order to elucidate the binding mode of the compound-A to proteasome, structure determination of the 20S proteosme complexed with compound-A was carried out. Isolated and concentrated 20S proteasome was co-crystallized with compound-A. Sitting drop vapor diffusion method was applied using 0.1M MES-NaOH (pH 6.5), 35% MPD, 50 mM magnesium acetate as a reservoir solution and 10 mg/ml proteasome and 20 mM compound-A as a sample solution. Crystals are isomorphous with the previous report [1]. Diffraction images were recorded at 100 K by using ADSC Quantum 210r CCD detector at NW12A of Photon Factory, Tsukuba, Japan. Initial phases were determined by molecular replacement method using the structure of the yeast 20S proteasome [1RYP] as a starting model and the structure model with a ligand was refined by using Refmac5 in CCP4 program package with an R value of 16.5% at 2.85 Å. The electron densities have been observed at the active site as mentioned above in the β-ring. Binding site of compound-A is closed to Tyr170 and Thr1 of the β5 subunit. The compound-A binds weakly to their residues by hydrogen bondings. It is quite different from the binding mode of the known potent proteasome inhibitor bortezomib [2].