3D Structure Elucidation of Thermostable L2 Lipase from Thermophilic Bacillus sp. L2

The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 × 0.1 × 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 Å and the crystal belongs to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 Å, α = β = γ = 90°. There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew’s coefficient of 2.85 Å Da⁻¹. The 3D structure of L2 lipase revealed topological organization of α/β-hydrolase fold consisting of 11 β-strands and 13 α-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca²⁺ and one Zn²⁺ were found in the L2 lipase molecule.     

Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P2₁2₁2₁, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.     

Capillary-Seeding Crystallization and Preliminary Crystallographic Analysis of a Solvent-Tolerant Elastase from Pseudomonas aeruginosa Strain K

Seeding is a versatile method for optimizing crystal growth. Coupling this technique with capillary counter diffusion crystallization enhances the size and diffraction quality of the crystals. In this article, crystals for organic solvent-tolerant recombinant elastase strain K were successfully produced through microseeding with capillary counter-diffusion crystallization. This technique improved the nucleation success rate with a low protein concentration (3.00 mg/mL). The crystal was grown in 1 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic dihydrate pH 5.6. The optimized crystal size was 1 × 0.1 × 0.05 mm³. Elastase strain K successfully diffracted up to 1.39 Å at SPring-8, Japan, using synchrotron radiation for preliminary data diffraction analysis. The space group was determined to be monoclinic space group P12₁1 with unit cell parameters of a = 38.99 Ǻ, b = 90.173 Å and c = 40.60 Å.     

Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme ��-d-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis

Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 × 250 × 375 μm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 × 40 × 60 μm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achived full size within 5–14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P4₃2₁2 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57–1.95 Å and the cell dimensions were between a = b = 99.2–100.8 Å and c = 183.2–183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4–3.5 ų·Da⁻¹ and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.     

Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T_m for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T_m as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.     

Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis

The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an αβγ heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 Å and belong to the C2 space group, with cell parameters a = 109.42 Å, b = 78.08 Å, c = 151.77 Å, β = 99.77°, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an αβγ heterotrimer.     

Synthesis, structure, and photovoltaic property of a nanocrystalline 2H perovskite-type novel sensitizer (CH3CH2NH3)PbI3

A new nanocrystalline sensitizer with the chemical formula (CH₃CH₂NH₃)PbI₃ is synthesized by reacting ethylammonium iodide with lead iodide, and its crystal structure and photovoltaic property are investigated. X-ray diffraction analysis confirms orthorhombic crystal phase with a = 8.7419(2) Å, b = 8.14745(10) Å, and c = 30.3096(6) Å, which can be described as 2 H perovskite structure. Ultraviolet photoelectron spectroscopy and UV-visible spectroscopy determine the valence band position at 5.6 eV versus vacuum and the optical bandgap of ca. 2.2 eV. A spin coating of the CH₃CH₂NH₃I and PbI₂ mixed solution on a TiO₂ film yields ca. 1.8-nm-diameter (CH₃CH₂NH₃)PbI₃ dots on the TiO₂ surface. The (CH₃CH₂NH₃)PbI₃-sensitized solar cell with iodide-based redox electrolyte demonstrates the conversion efficiency of 2.4% under AM 1.5 G one sun (100 mW/cm²) illumination.     

Synthesis, Structure of Nitrogen-Containing Phosphinogold(I) Ferrocenes. In vitro Activity against Bladder and Colon Carcinoma Cell Lines

The gold salt [(tht)AuCl] was reacted with [1-N,N-dimethylaminométhyl-2-diphenylphosphino]ferrocene (1) forming the bimetallic derivative 4. The reaction of methyl iodide and tetramethylammonium bromide on the chloride 4 produced the ammonium salt 5 and the bromide 6 respectively. New aminophosphines 2 and 3, which represent two of the rare phosphorylated metallocenes containing P(III)-N bond have also been coordinated to gold(I) to form 7 and 8. The presence of the ethoxy group in 7 provides evidence for the lability of one nitrogen-phosphorus bond. The X-ray structure of compounds 4 and 7 have been established. Both crystallize in space group P2₁/c, monoclinic, with a = 11.095(2) Å, b = 12.030(3) Å, c = 17.763(4) Å, β= 94.02(2)∘, Z = 4 for 4 and a = 14.863(3) Å, b = 8.036(5)Å, c = 18.062(5)Å, β =101.64(1)°, Z = 4 for 7. ¹⁹⁷Au Mössbauer data are in good agreement with those for other linear P-Au-Cl containing complexes. The compounds were evaluated for in vitro anti-tumour activity against two human tumours. Differential cytotoxicity was observed with activity comparable to cisplatin, with the exception of one compound which was significantly more cytotoxic.     

Antibacterial activity of silver-doped hydroxyapatite nanoparticles against gram-positive and gram-negative bacteria

Ag-doped nanocrystalline hydroxyapatite nanoparticles (Ag:HAp-NPs) (Ca_10-xAg_x(PO₄)₆(OH)₂, x_Ag = 0.05, 0.2, and 0.3) with antibacterial properties are of great interest in the development of new products. Coprecipitation method is a promising route for obtaining nanocrystalline Ag:HAp with antibacterial properties. X-ray diffraction identified HAp as an unique crystalline phase in each sample. The calculated lattice constants of a = b = 9.435 Å, c = 6.876 Å for x_Ag = 0.05, a = b = 9.443 Å, c = 6.875 Å for x_Ag = 0.2, and a = b = 9.445 Å, c = 6.877 Å for x_Ag = 0.3 are in good agreement with the standard of a = b = 9.418 Å, c = 6.884 Å (space group P6₃/m). The Fourier transform infrared and Raman spectra of the sintered HAp show the absorption bands characteristic to hydroxyapatite. The Ag:HAp nanoparticles are evaluated for their antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae, Providencia stuartii, Citrobacter freundii and Serratia marcescens. The results showed that the antibacterial activity of these materials, regardless of the sample types, was greatest against S. aureus, K. pneumoniae, P. stuartii, and C. freundii. The results of qualitative antibacterial tests revealed that the tested Ag:HAp-NPs had an important inhibitory activity on P. stuartii and C. freundii. The absorbance values measured at 490 nm of the P. stuartii and C. freundii in the presence of Ag:HAp-NPs decreased compared with those of organic solvent used (DMSO) for all the samples (x_Ag = 0.05, 0.2, and 0.3). Antibacterial activity increased with the increase of x_Ag in the samples. The Ag:HAp-NP concentration had little influence on the bacterial growth (P. stuartii).     

Structural studies of poly[(2S,3S)-2,3-dimethoxybutylene alkanamide]s

The structural characterization of a series of stereoregular polyamides 4,n (n=8, 10, 12), which were obtained from polycondensation of (2S,3S)-2,3-dimethoxy-1,4-butanediamine with aliphatic dicarboxylic acids, has been carried out. X-ray diffraction of powder and fiber samples were analyzed together with electron diffraction patterns of single crystals obtained by isothermal crystallization in solution. Experimental results based on lattice parameters data were used to build a preliminary crystal model using the Cerius program. The main characteristic of the proposed model is the presence of intermolecular hydrogen bonds along the crystal a direction, similarly arranged to conventional nylons.     

One-Dimensional Hydrogen-Bonded Infinite Chain from Nickel(II) Tetraaza Macrocyclic Complex and 1,2-Cyclopentanedicarboxylate Ligand

The reaction of [Ni(L)]Cl₂·2H₂O (L = 3,14-dimethyl-2,6,13,17-tetraazatricyclo [14,4,0^1.18,0^7.12]docosane) with trans-1,2-cyclopentanedicarboxylic acid (H₂-cpdc) yields a 1D hydrogen-bonded infinite chain with formula [Ni(L)(H-cpdc⁻)₂] (1). This complex has been characterized by X-ray crystallography, spectroscopy and cyclic voltammetry. The crystal structure of 1 exhibits a distorted octahedral geometry about Ni atom with four nitrogen atoms of the macrocycle and two oxygen atoms of the H-cpdc⁻ ligand at the axial position. Compound 1 crystallizes in the monoclinic system P2₁/c with a = 8.7429(17), b = 10.488(2), c = 18.929(4) Å, β = 91.82(2), V = 1734.8(6) ų, Z = 2. Electronic spectrum of 1 reveals a high-spin octahedral environment. Cyclic voltammetry of 1 undergoes two waves of a one-electron transfer corresponding to Ni^II/Ni^III and Ni^II/Ni^I processes.     

Crystal Structure of Dimeric Flavodoxin from Desulfovibrio gigas Suggests a Potential Binding Region for the Electron-Transferring Partner

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.     

Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD)

Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3₁21 (or its enantiomorph P3₂21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 Å and γ = 120°. The crystals were obtained at room temperature and diffracted to 2.0 Å resolution.     

Effects of sintering aid and atmosphere powder on the growth of (K0.5Na0.5)NbO3 single crystals fabricated by solid-state crystal growth method

(K_0.5Na_0.5)NbO₃ (KNN) + x (= 1, 0.5, 0.05, and 0) wt%Co₃O₄ single crystals were fabricated by a solid-state crystal growth method with a KTaO₃ seed crystal and a KNN atmosphere powder, and the effects of the sintering aid content x and the addition of Co₃O₄ to the atmosphere powder on the growth of the single crystals were investigated. The formation of pores in the single crystals was suppressed by a decrease of x, which, however, decreased the crystal growth length. On the other hand, dense and large KNN single crystals could be fabricated by sintering with a KNN + 5 wt%Co₃O₄ atmosphere at x = 0. The dielectric, ferroelectric, and piezoelectric properties of the KNN single crystals were comparable to those of reported (K,Na)NbO₃ single crystals. These results would be useful for fabricating dense and large single crystals by the solid-state crystal growth method.     

Crystal structures of poly(n-methylen-di-O-methyl-l-tartaramide)s with n=3, 5, 7 and 9

The structural characterization of poly(n-methylen-di-O-methyl-l-tartaramide)s with n=3, 5, 7 and 9 has been carried out using optical microscopy, thermal analysis, X-ray diffraction and electron microscopy. X-ray diffraction of powder and fiber samples were analyzed together with electron diffraction patterns of single crystals obtained from isothermal crystallization in solution. Experimental results based on crystallographic data were used to build a crystal model using the Cerius program. This model is based in chain packing with an arrangement of hydrogen-bonded sheets, being the resultant crystal structure similar to the exhibited in conventional polyamides.     

Interaction of BSA protein with copper evaluated by electrochemical impedance spectroscopy and quartz crystal microbalance

The interaction of bovine serum albumin (BSA) protein with copper in phosphate buffer solution has been studied by a combination of electrochemical impedance spectroscopy (EIS) close to the open circuit potential, with simultaneous monitoring by the electrochemical quartz crystal microbalance (EQCM), in order to throw light on BSA adsorption. Copper films were electroplated onto gold quartz crystals and mounted in the EQCM. Experiments were conducted in the presence and absence of dissolved oxygen and of BSA and the results show the influence of O₂ on the protein/metal interaction and also show specific interactions between BSA and copper. The good reproducibility obtained in these experiments suggests future application to other systems and which should lead to a better understanding of the use of such types of protein as corrosion inhibitors.     

TupA: A Tungstate Binding Protein in the Periplasm of Desulfovibrio alaskensis G20

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P2₁ space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.     

Surface Engineering of Top7 to Facilitate Structure Determination

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/R_free values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.     

1,4-二硝基咪唑的制备与晶体结构

以咪唑作为原料,经两步反应,得到1,4-二硝基咪唑,并于室温下培养出单晶。通过X射线单晶结构分析法测定其晶体结构。结果表明,晶体属于正交晶系,空间群为P212121,晶体学参数为：a=5.832 3（13）nm,b=9.562（2）nm,c=10.302（2）nm,V=574.5（2）nm3,Z=4,DC=1.828 g/cm3,F（000）=320。     

Shape-controlled fabrication of polypyrrole microstructures by replicating organic crystals through electrostatic interactions

Polypyrrole (PPy) microstructures with diverse shapes were synthesized in an aqueous inorganic salt medium including organic crystals and pyrrole (Py). A series of sulfobenzoic acid salt forms with various cations (K⁺, Na⁺, Li⁺, NH₄⁺) in different positions (para, meta, ortho) of the sulfonate group on the benzene ring were used to form organic crystals as sacrificial templates. Using these crystals, we produced five different shapes of PPy microstructures (hexagonal microplates, curled nanofibers, lozenge-shaped microplates, rigid rods, parallelogram microplates), which replicated the shapes of the organic crystal templates through electrostatic interaction between the anionic crystal surfaces and the cationic PPy chains. In contrast, PPy that was polymerized without crystals showed bulky agglomerates of 200-500 nm size. The electrical properties were dictated by the molecular structures of the organic salt molecules used. While the highest conductivity (200.3 Scm⁻¹) was observed in PPy using crystals of para-linked 4-sulfobenzoic acid monopotassium salt, the lowest conductivity (0.8 Scm⁻¹) was observed in PPy prepared in the presence of crystals of ortho-linked 2-sulfobenzoic acid monoammonium salt.