Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 A resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 A. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 57%.
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09×0.19×0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 Å, β=96.40°.
Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.
TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces 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 in Escherichia coli and 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 group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20,000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 A resolution and data were collected to 99.6% completeness at 8.9 A. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 A. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.
The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 A resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation.
The title compound, Ph3(PhCH2)P(+)·Cl(-)·H2O, was obtained unintentionally as the product of an attempted synthesis of a silver di-thio-carbamate complex using benzyl-tri-phenyl-phospho-nium as the counter-ion. The asymmetric unit consists of a phospho-nium cation and a chloride anion, and a water mol-ecule of crystallization. In the crystal, the chloride ion is linked to the water mol-ecule by an O-H⋯Cl hydrogen bond. The three units are further linked via C-H⋯Cl and C-H⋯O hydrogen bonds and C-H⋯ π inter-actions, forming a three-dimensional structure.
A new polymorphic form of the title compound, C8H8O3, is described in the centrosymmetric monoclinic space group P21/c with Z' = 1 as compared to the first polymorph, which crystallizes with two conformers (Z' = 2) in the asymmetric unit in the same space group. In the crystal of the second polymorph, inversion dimers linked by O-H⋯O hydrogen bonds occur and these are linked into zigzag chains, propagating along the b-axis direction by C-H⋯O links. The crystal structure also features a weak π-π inter-action, with a centroid-to-centroid distance of 3.8018 (6) Å. The second polymorph of the title compound is less stable than the reported first polymorph, as indicated by its smaller calculated lattice energy.
A truncated form of an α-amylase, GTA, from thermophilic Geobacillus thermoleovorans CCB_US3_UF5 was biochemically and structurally characterized. The recombinant GTA, which lacked both the N- and C-terminal transmembrane regions, functioned optimally at 70°C and pH 6.0. While enzyme activity was not enhanced by the addition of CaCl2, GTA's thermostability was significantly improved in the presence of CaCl2. The structure, in complex with an acarbose-derived pseudo-hexasaccharide, consists of the typical three domains and binds one Ca(2+) ion. This Ca(2+) ion was strongly bound and not chelated by EDTA. A predicted second Ca(2+)-binding site, however, was disordered. With limited subsites, two novel substrate-binding residues, Y147 and Y182, may help increase substrate affinity. No distinct starch-binding domain is present, although two regions rich in aromatic residues have been observed. GTA, with a smaller domain B and several shorter loops compared to other α-amylases, has one of the most compact α-amylase folds that may contribute greatly to its tight Ca(2+) binding and thermostability.
A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.
The title compound, C16H10, crystallizes with four unique mol-ecules, designated 1-4, in the asymmetric unit of the monoclinic unit cell. None of the mol-ecules is planar, with the benzene rings of mol-ecules 1-4 inclined to one another at angles of 42.41 (4), 24.07 (6), 42.59 (4) and 46.88 (4)°, respectively. In the crystal, weak C-H⋯π(ring) interactions, augmented by even weaker C C-H⋯π(alkyne) contacts, generate a three-dimensional network structure with inter-linked columns of mol-ecules formed along the c-axis direction.
Piroxicam is a nonsteroidal anti-inflammatory drug with low aqueous solubility which exhibits polymorphism. The present study was carried out to develop polymorphs of piroxicam with enhanced solubility and dissolution rate by the crystal modification technique using different solvent mixtures prepared with PEG 4000 and PVP K30. Physicochemical characteristics of the modified crystal forms of piroxicam were investigated by X-ray powder diffractometry, FT-IR spectrophotometry and differential scanning calorimetry. Dissolution and solubility profiles of each modified crystal form were studied and compared with pure piroxicam. Solvent evaporation method (method I) produced both needle and cubic shaped crystals. Slow crystallization from ethanol with addition of PEG 4000 or PVP K30 at room temperature (method II) produced cubic crystal forms. Needle forms produced by method I improved dissolution but not solubility. Cubic crystals produced by method I had a dissolution profile similar to that of untreated piroxicam but showed better solubility than untreated piroxicam. Cubic shaped crystals produced by method II showed improved dissolution, without a significant change in solubility. Based on the XRPD results, modified piroxicam crystals obtained by method I from acetone/benzene were cube shaped, which correlates well with the FTIR spectrum; modified needle forms obtained from ethanol/methanol and ethanol/acetone showed a slight shift of FTIR peak that may be attributed to differences in the internal structure or conformation.
Refined bleached and deodorized palm oil (RBDPO) was crystallized from the melt in a thermally controlled water bath at 14 and 22°C for 90 min. Slurries were withdrawn after 5, 15, 30, 60 and 90 min of crystallization for crystal morphology studies. Crystallization was also performed in a similar manner using a Labmax reactor connected to a FBRM detector to obtain the information on crystal count and size distribution during crystallization. Based on the shape of the crystals viewed under the microscope, all crystals appeared as needle shaped spherulitic β´- form at both crystallization temperatures studied. Crystals were slightly larger with increase in crystallization time and at higher crystallization temperature (22°C). Crystals size range between 4.34 to 22.29µm. FBRM recorded high count of crystals with increased in crystallization time and at lower temperature (14°C).
Diacylglycerol at 1 or 6% was added into refined bleached and deodorized palm oil (RBDPO) and crystallized from the melt in a thermally controlled water bath at 22°C for 90 min. Slurries were withdrawn after 5, 15, 30, 60 and 90 min of crystallization for solid fat content (SFC) and crystal morphology studies. Crystallization was also performed in a similar manner using a Labmax reactor connected to a FBRM detector to obtain the information on crystal count and size distribution during crystallization. SFC of the slurries increased with increase in crystallization time up to a certain level followed by a plateau. SFC of RBDPO added with DAG was also higher with the increase in percentage of DAG added and no induction time was observed to initiate crystallization in RBDPO added with DAG. The addition of DAG caused rapid crystallization of RBDPO as observed by enhance nucleation and larger crystal size with increase in the percentages of DAG added.
There are a number of differential diagnoses for crystal deposits in the cornea. With the presence of a corneal epithelial defect, the differential diagnosis can be narrowed down to either infective causes or deposits from topical medications. This report describes a case of crystal deposits in the cornea from the use of Vitamin C eye drops.
White rice husk ash (RHA), an agriculture waste containing crystalline tridymite and alpha-cristobalite, was used as a silica source for zeolite Beta synthesis. The crystallization of zeolite Beta from RHA at 150 degrees C in the presence of tetraethylammonium hydroxide was monitored by XRD, FTIR and (29)Si MAS NMR techniques. It was found that zeolite Beta started to form after 12h and the complete crystallization of zeolite Beta phase was achieved after 2d. XRD, (29)Si MAS NMR and solid yield studies indicate that the transformation mechanism of silica present in RHA to zeolite Beta involves dissolution of the ash, formation of an amorphous aluminosilicate after 6h of crystallization, followed by dissolution in the mother liquor and final transformation to pure zeolite Beta crystals.
Metanilate-layered double hydroxide nanohybrid compound was synthesized for controlled release purposes through co-precipitation method of the metal cations and organic anion. The effect of various divalent metal cations (M2+), namely Zn2+, Mg2+ and Ca2+ on the formation of metanilate-LDH nanohybrids, in which metanilate anion was intercalated into three different layered double hydroxide (LDH) systems; Zn-Al, Mg-Al and Ca-Al were investigated. The syntheses were carried out with M2+ to Al3+ initial molar ratio, R of 4. The pH of the mother liquor was maintained at pH 7.5 and 10 during the synthesis, and the resulting mixture was aged at around 70 degrees C for about 18 h. The intercalation of metanilate anion into the host was found to be strongly influenced by the M2+ that formed the inorganic metal hydroxide layers. Under our experimental condition, the formation of the nanohybrid materials was found to be more feasible for the Zn-Al than for the other two systems, in which the former showed well-ordered layered organic-inorganic nanohybrid structure with good crystallinity. Intercalation is confirmed by the expansion of the interlayer spacing to about 15-17 A when metanilate was introduced into the interlamellae of Zn-Al LDHs. In addition, CHNS and FTIR analyses also support that metanilate anion has been successfully intercalated into the interlamellae of the inorganic LDH. Apart from M2+, this study also shows that the initial pH of the mother liquor plays an important role in determining the physicochemical properties of the resulting nanohybrids, especially the mole fraction of the Zn2+ substituted by the Al3+ ion in the LDH inorganic sheets which in turn controlled the loading percentage of the organic anion, surface properties and the true density. Preliminary study shows that LDH can be used to host beneficial guests, active agent with controlled release capability of the guests. Generally the overall process is governed by pseudo second order kinetic but for the first 180 min, the release process can be slightly better described by parabolic diffusion than the other models.
Microporous polymeric matrices prepared from poly(ɛ-caprolactone) [PCL] were evaluated for controlled vaginal delivery of the antiprotozoal agent (tinidazole) in the treatment of the sexually transmitted infection, trichomoniasis. The matrices were produced by rapidly cooling co-solutions of PCL and tinidazole in acetone to -80 °C to induce crystallisation and hardening of the polymer. Tinidazole incorporation in the matrices increased from 1.4 to 3.9% (w/w), when the drug concentration in the starting PCL solution was raised from 10 to 20% (w/w), giving rise to drug loading efficiencies up to 20%. Rapid 'burst release' of 30% of the tinidazole content was recorded over 24 h when the PCL matrices were immersed in simulated vaginal fluid. Gradual drug release occurred over the next 6 days resulting in delivery of around 50% of the tinidazole load by day 7 with the released drug retaining antiprotozoal activity at levels almost 50% that of the 'non-formulated' drug in solution form. Basic modelling predicted that the concentration of tinidazole released into vaginal fluid in vivo from a PCL matrix in the form of an intravaginal ring would exceed the minimum inhibitory concentration against Trichomonas vaginalis. These findings recommend further investigation of PCL matrices as intravaginal devices for controlled delivery of antiprotozoal agents in the treatment and prevention of sexually transmitted infections.
The rational design of a glycolipid application (e.g. drug delivery) with a tailored property depends on the detailed understanding of its structure and dynamics. Because of the complexity of sugar stereochemistry, we have undertaken a simulation study on the conformational dynamics of a set of synthetic glycosides with different sugar groups and chain design, namely dodecyl β-maltoside, dodecyl β-cellobioside, dodecyl β-isomaltoside and a C12C10 branched β-maltoside under anhydrous conditions. We examined the chain structure in detail, including the chain packing, gauche/trans conformations and chain tilting. In addition, we also investigated the rotational dynamics of the headgroup and alkyl chains. Monoalkylated glycosides possess a small amount of gauche conformers (∼20%) in the hydrophobic region of the lamellar crystal (LC) phase. In contrast, the branched chain glycolipid in the fluid Lα phase has a high gauche population of up to ∼40%. Rotational diffusion analysis reveals that the carbons closest to the headgroup have the highest correlation times. Furthermore, its value depends on sugar type, where the rotational dynamics of an isomaltose was found to be 11-15% and more restrained near the sugar, possibly due to the chain disorder and partial inter-digitation compared to the other monoalkylated lipids. Intriguingly, the present simulation demonstrates the chain from the branched glycolipid bilayer has the ability to enter into the hydrophilic region. This interesting feature of the anhydrous glycolipid bilayer simulation appears to arise from a combination of lipid crowding and the amphoteric nature of the sugar headgroups.