High Solvent Content Research Articles

Structure determination of VEGF-E by sulfur SAD

The crystal structure of VEGF-E was solved by the sulfur single-wavelength anomalous dispersion method (S-SAD) using highly redundant low-resolution data collected at a wavelength of lambda approximately 1.7 A with an estimated anomalous signal of 1.5%. 11 sulfur sites, nine out of 16 disulfide bonds and two out of 12 methionines could be located in the asymmetric unit using data truncated at a resolution of 4.1 A; however, none of the common diffraction data-quality indicators for SAD allowed clear discrimination between successful and unsuccessful resolution cutoffs. The high solvent content of 75% allowed efficient density modification to be performed and an unbiased electron-density map of good quality to be generated. This study demonstrates the strength of S-SAD for phasing using accurate highly redundant data at low resolution.

Structure of the Unphosphorylated STAT5a Dimer*

STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-A crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their beta-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells.

Crystallization and preliminary X-ray diffraction studies of the WW4 domain of the Nedd4-2 ubiquitin–protein ligase

Ubiquitin-mediated protein modification via covalent attachment of ubiquitin has emerged as one of the most common regulatory processes in all eukaryotes. Nedd4-2, closely related to neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4), is a multimodular ubiquitin-protein ligase comprised of four WW domains and a Hect domain. The WW domains recognize the proline-rich motifs on the multi-subunit amiloride-sensitive epithelial sodium channel (ENaC). To gain insights into the binding of the WW domain to proline-rich peptides, a protein fragment (78 amino acids) containing the fourth WW domain (WW4) of the Nedd4-2 protein was purified and crystallized and X-ray diffraction data were collected. A data set was obtained to 2.5 A resolution from a cryocooled single crystal at a synchrotron source. The crystals belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 113.43, c = 103.21 A. Analysis of the self-rotation function suggests the presence of four WW4 molecules in the asymmetric unit, with a high unit-cell solvent content of 74%.

The incorporation of large impurities into virus crystals

Virus crystals can incorporate a wide range of unusual impurities, not possible for conventional crystals, or even most protein crystals because of the large size of their constituent particles. These impurities include anomalous virions, satellite viruses and biological fibers. Examples of several of these unusual impurities are presented here, along with some of the consequences for the crystal lattices. The high solvent content, the forgiving character of the lattice and the plasticity of the virions allow these incorporations to be possible.

Ultra-deep x-ray lithography of densely packed SU-8 features: I. An SU-8 casting procedure to obtain uniform solvent content with accompanying experimental results

Although the benefits of SU-8 as a thick photoresist material for MEMS applications have been shown by various researchers, its use for ultra-thick applications (>1 mm) has been hampered by the difficulties associated with the high solvent gradient of the resultant layers. In this paper, the lithographic performance of the traditional methods of thick resist formation based on the casting of high solvent content SU-8 is presented. As an extension of the work, a new photoresist preparation method called ‘dry chip casting’ was developed. The new method involves the reduction of the solvent content of a given photoresist until the desired content is reached, the collection of the ‘dry’ chips and re-constitution on a substrate with the aid of heat and vacuum. The dry chip casting method yielded average solvent contents of about 7% (±0.5%) throughout the thickness of the SU-8 layers, with excellent x-ray lithographic characteristics. Results of ultra-deep x-ray lithography experiments in SU-8 layers as thick as 3 mm are presented in this paper.

Purification, crystallization and preliminary X-ray diffraction studies of the archaeal virus resolvase SIRV2

The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8, b = 99.9, c = 87.6, beta = 109.46 degrees, and a full data set has been collected at 3.4 A resolution. The self-rotation function indicates the presence of two dimers in the asymmetric unit and a high solvent content (77%). Molecular-replacement trials using known similar resolvase structures have so far been unsuccessful, indicating possible significant structural rearrangements.

Influence of surfactant addition sequence on the suspension properties and electrophoretic deposition behaviour of alumina and zirconia

A well-known polyelectrolyte salt, ammonium polymethacrylate (Darvan-C) is used to stabilise ethanol-based Al 2O 3 and Ce-ZrO 2 suspensions with butylamine addition. The sequence in which n-butylamine and Darvan-C are added to the suspension greatly affects the properties of the wet deposit obtained by electrophoretic deposition. To investigate this effect, electrical conductivity of the suspension and the shear rate dependence of its viscosity are investigated. When n-butylamine is added first, the equilibrium in the suspension is almost immediately reached and a plastically deformable wet deposit is obtained over a large n-butylamine/Darvan-C ratio. The suspension has a shear-thinning viscosity and the deformable deposit is characterised by a high solvent content, which allows the rearrangement of particles during drying. When Darvan-C is added before the n-butylamine, the wet deposit is smooth and rigid. The suspension has a lower viscosity and a near-Newtonian behaviour is observed. A similar behaviour is observed for Al 2O 3 and Ce-ZrO 2 suspensions. The green density of the dried deposits is not influenced by the addition sequence and higher green densities are obtained for Al 2O 3 when compared to Ce-ZrO 2.

Crystallization and preliminary X-ray diffraction analysis of human growth and differentiation factor 5 (GDF-5)

Growth and differentiation factor 5 (GDF-5) belongs to the large TGF-beta superfamily of secreted signalling proteins and plays a pivotal role in skeletal development during embryogenesis. The gene for human GDF-5 was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained that diffracted to 2.2 A resolution. A native data set was acquired, showing that the crystals belong to a trigonal space group, i.e. P3(1)21 or P3(2)21, with unit-cell parameters a = b = 97.1, c = 48.3 A. Initial analysis suggest the presence of only one monomer in the asymmetric unit, resulting in a high solvent content of 72% in the crystal.

Crystallization and preliminary X-ray crystallographic analysis of agkicetin-C fromDeinagkistrodon acutusvenom

The crystallization and preliminary crystallographic analysis of agkicetin-C, a well known platelet glycoprotein Ib (GPIb) antagonist from the venom of Deinagkistrodon acutus found in Anhui Province, China is reported. Crystals of agkicetin-C suitable for structure determination were obtained from 1.8 M ammonium sulfate, 40 mM MES pH 6.5 with 2%(v/v) PEG 400. Interestingly, low buffer concentrations of MES seem to be necessary for crystal growth. The crystals of agkicetin-C belong to space group C2, with unit-cell parameters a = 177.5, b = 97.7, c = 106.8 A, beta = 118.5 degrees, and diffract to 2.4 A resolution. Solution of the phase problem by the molecular-replacement method shows that there are four agkicetin-C molecules in the asymmetric unit, with a VM value of 3.4 A3 Da(-1), which corresponds to a high solvent content of approximately 64%. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that may represent additional elements of non-crystallographic symmetry.

Two orthorhombic crystal structures of a galactose-specific lectin from Artocarpus hirsuta in complex with methyl-alpha-D-galactose.

Based on their carbohydrate specificity, the jacalin family of lectins can be divided into two groups: galactose-specific and mannose-specific. The former are cytoplasmic proteins, whereas the latter are localized in the storage vacuoles of cells. It has been proposed that the post-translational modification in some of the lectins that splits their polypeptide chains into two may be crucial for galactose specificity. The mannose-specific members of the family are single-chain proteins that lack the above modification. Although the galactose-specific and the mannose-specific jacalin-type lectins differ in their sequences, they share a common fold: the beta-prism I fold, which is characteristic of Moraceae plant lectins. Here, two crystal structures of a jacalin-related lectin from Artocarpus hirsuta, which is specific for galactose, in complex with methyl-alpha-D-galactose are reported. The lectin crystallized in two orthorhombic forms and one hexagonal form under similar conditions. The crystals had an unusually high solvent content. The structure was solved using the molecular-replacement method using the jacalin structure as a search model. The two orthorhombic forms were refined using data to 2.5 and 3.0 A resolution, respectively. The structures of the A. hirsuta lectin and jacalin are identical. In orthorhombic form I the crystal packing provides three different micro-environments for sugar binding in the same crystal. The observed difference in the specificity for oligosaccharides between the A. hirsuta lectin and jacalin could only be explained based on differences in the molecular associations in the packing and variation of the C-terminal length of the beta-chain. The observed insecticidal activity of A. hirsuta lectin may arise from its similar fold to domain II of the unrelated delta-endotoxin from Bacillus thuringiensis.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl.

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 A resolution, have unit-cell parameters a = b = 179.92, c = 185.82 A and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

In-house phase determination of the lima bean trypsin inhibitor: a low-resolution sulfur-SAD case.

SAD (single-wavelength anomalous diffraction) has enormous potential for phasing proteins using only the anomalous signal of the almost ubiquitous native sulfur, but requires extremely precise data. The previously unknown structure of the lima bean trypsin inhibitor (LBTI) was solved using highly redundant data collected to 3 A using a CCD detector with a rotating-anode generator and three-circle goniometer. The seven 'super-S' atoms (disulfide bridges) were located by dual-space recycling with SHELXD and the high solvent content enabled the density-modification program SHELXE to generate high-quality maps despite the modest resolution. Subsequently, a 2.05 A synchrotron data set was collected and used for further phase extension and structure refinement.

Synthesis of π-Conjugated Organometallic Polymer Networks

The present paper reports the synthesis of π-conjugated organometallic polymer networks based on poly[2,5-diotyloxy-1,4-diethynyl-phenylene-alt-2,5,-bis-(2'-ethylhexyloxy)-1,4-phenylene] (EHO-OPPE), a soluble poly(p-phenylene ethynylene) (PPE) derivative. The ethynylene moities of the polymer coordinate to Pt 0 centers, which act as conjugated cross-links. These materials are readily accessible through ligand-exchange reactions between the linear PPE and Pt(styrene) 3 . The in-situ NMR investigation of model reactions of Pt(styrene) 3 with diphenylacetylene (DPA) has shown that the ethynylene moieties comprised in the PPE readily coordinate to Pt 0 centers under release of the relatively weakly-bound styrene ligands. Spin-coating has resulted in cross-linked films of good optical quality. We also have been able to produce PPE-Pt-gels with high solvent content (>95 wt.-%). As expected, the coordination of Pt markedly influences the photophysical characteristics of the pPE. The photoluminescence is efficiently quenched, and the absorption maximum in the visible regime experiences a hypsochromic shift

Emulsion stability based on phase behavior in sodium naphthenates containing systems: Gels with a high organic solvent content

Addition of heptane to a sodium naphthenates/toluene/water system at 25 °C reduces the lamellar liquid-crystal phase range and increases the microemulsion phase range. Both of these effects result in the extension of the composition range where emulsions have low stability. This effect is even stronger at 40 °C. Heptane addition also results in the formation of very stable emulsions within the overlapping phase-existence ranges of aqueous (L1) and organic (L2) phases. Stable non-birefringent gel observed in equilibrium with L1 and L2 phases contains only a small percentage of water and sodium naphthenates. The swelling behavior of an unstable gel, an emulsion previously compressed by centrifugation, appears to be due to a stepwise thickening of the thin liquid films between the droplets.

Macromolecular phasing with SHELXE

Abstract SHELXE was designed to provide a simple, fast and robust route from substructure sites found by the program SHELXD to an initial electron density map, if possible with an indication as to which heavy-atom enantiomorph is correct. This should be understood as a small contribution to high-throughput structural genomics. The new sphere of influence algorithm combined with a fuzzy solvent boundary enables some chemical knowledge to be incorporated into the density modification in a general and effective manner. In the special cases of high solvent content (greater than 0.6) or very high resolution data (better than 1.5 Å) high quality maps can be produced. This raises the possibility of a new paradigm for atomic resolution structure refinement: instead of alternating atom parameter refinement with weighted electron density maps calculated with the phases of the current model, which inevitably leads to some model bias, all model building should be based on the model free experimental density map!

Morphology of crosslinked poly(butyl methacrylate-co-methyl methacrylate) porous membranes

For the separation of liquid mixtures, IPN structure could be used to enhance the mechanical strength and the selectivity of membrane by restricting the swelling of the transporting polymer component networks. In this study, for the separation of water–ethanol mixture, composite membranes having polyacrylic acid–poly(butyl methacrylate-co-methyl methacrylate) interpenetrating polymer network (PAA–P(BMA-co-MMA) IPN) skin layer supported on crosslinked and porous poly(BMA-co-MMA) were prepared. The porous x-P(BMA-co-MMA) sublayer was formed by polymerizing monomer mixture in the presence of solvent and followed by immersion precipitation. Morphology changes and the factors involved in the pore formation of the crosslinked porous sublayers were investigated. Final morphology of the sublayer revealed three types of morphology depending on the amount of added solvent and degree of crosslinking: (1) non-porous structure with higher degree of crosslinking; (2) porous cellular structure with lower degree of crosslinking and (3) finger-like structure with lower degree of crosslinking and higher solvent content. We could obtain crosslinked highly porous interconnected poly(BMA-co-MMA) sulayer by polymerizing monomer mixture with 70% NMP and M ̄ C =40 ,000. It was found that crosslinked porous structure could be obtained when phase separation occurred before gelation. The thickness of the hydrophilic (PAA)–hydrophobic (P(BMA-co-MMA)) IPN skin layer of the composite membrane was about 10 μm. For the azeotropic water–ethanol mixture (95 wt.% ethanol), the PAA 2–P(BMA15-co-MMA15) 40 IPN composite membrane had a separation factor of 114 and a permeation rate of 160 g/m 2 h at 25 °C.

Structure of a domain-opened mutant (R121D) of the human lactoferrin N-lobe refined from a merohedrally twinned crystal form.

Human lactoferrin is an iron-binding protein with a bilobal structure. Each lobe contains a high-affinity binding site for a single Fe(3+) ion and an associated CO(3)(2-) ion. Although iron binds very tightly, it can be released at low pH, with an accompanying conformational change in which the two domains move apart. The Arg121Asp (R121D) mutant of the N-lobe half-molecule of human lactoferrin was constructed in order to test whether the Asp121 side chain could substitute for the CO(3)(2-) ion at the iron-binding site. The R121D mutant protein was crystallized in its apo form as it lost iron during crystallization. The crystals were also merohedrally twinned, with a twin fraction close to 0.5. Starting from the initial molecular-replacement solution [Breyer et al. (1999), Acta Cryst. D55, 129-138], the structure has been refined at 3.0 A resolution to an R factor of 13.9% (R(free) of 19.9%). Despite the moderate resolution, the high solvent content and non-crystallographic symmetry contributed to electron-density maps of excellent quality. Weakened iron binding by the R121D mutant is explained by occlusion of the anion-binding site by the Asp side chain. The opening of the two domains in the apoR121D structure (a rotation of 54 degrees ) closely matches that of the N-lobe in full-length lactoferrin, showing that the extent of the conformational change depends on properties inherent to the N-lobe. Differences in the C-terminal portion of the N-lobe (residues 321-332) for apoR121D relative to the closed wild-type iron-bound structure point to the importance of this region in stabilizing the open form.

De novo phasing of two crystal forms of tryparedoxin II using the anomalous scattering from S atoms: a combination of small signal and medium resolution reveals this to be a general tool for solving protein crystal structures.

The de novo phasing of the structures of two crystal forms of tryparedoxin II from Crithidia fasciculata has been carried out using single-wavelength anomalous diffraction techniques exploiting only the small anomalous signal from the S atoms intrinsic to the native protein. Data were collected at 1.77 A wavelength, where the Bijvoet ratio is approximately 1.2%. Data collected to d(min) = 2.5 A from a crystal of form I, which has a diffraction limit of d(min) = 1.5 A and a solvent content of approximately 46%, produced readily interpretable electron-density maps. When these phases were extended to the resolution limit of the crystals, almost the entire model could be traced automatically. Crystals of form II have a much higher solvent content, approximately 72%, and a much lower diffraction limit than form I and at 1.77 A wavelength yielded data only to d(min) = 2.7 A. Despite the medium resolution of the data for this crystal form, it was possible both to determine the heavy-atom partial structure and then use it to produce, still at d(min) = 2.7 A, an excellent quality interpretable electron-density map. This was then improved by phase extension to the d(min) = 2.35 A diffraction limits of a different crystal for which data were collected on a more intense beamline. The success of this latter structure solution markedly increases the potential use in macromolecular crystal structure determination of the anomalous signal available from S atoms that occur naturally in proteins and, as is discussed, has significant implications for structure determination in the high-throughput era.

Characterization of solvent—filler-poly(dimethylsiloxane) membranes by Differential Scanning Calorimetry

The properties of three polydimethylsiloxane (PDMS) membranes filled with crystalline zeolites and with amorphous SiO2, respectively, before and after sorption of organic solvents were studied by DSC. The PDMS crystallinity of the membranes with sorbed solvents is affected by both the solvent and the filler natures. For SiO2 -filled membrane, the PDMS crystallinity increases first due to the weakening of the physical crosslinks by the solvent. In the case of zeolite fillers, the PDMS crystallinity decreases first with the increase in the solvent content due to a decrease in the zeolite-induced crystallization effect by the solvent adsorption on zeolites. At high solvent contents, the solvent influence on the PDMS crystallite prevails. A medium solvent (ethyl acetate) induces the polymer crystallization, while a good solvent (cyclohexane) disrupts PDMS crystallites.

Polyacrylonitrile–propylene carbonate–CuX 2 (X=Cl, Br, CF 3SO 3) solid polymer electrolyte

A series of poly(acrylonitrile)–propylene carbonate–CuX 2 (PAN–PC–CuX 2, where X=Cl, Br and CF 3SO 3) foils of thickness between 0.03 and 0.09 cm, were prepared by means of the solution cast technique. The PAN–PC–CuX 2 system forms flexible foils of good mechanical properties even at high solvent and salt contents. Conductivities of the foils obtained from the analysis of impedance spectra, reach the level of ca. 5×10 −3 Ω −1 cm −1. The temperature variation of the conductivity for all the systems studied is of the Arrhenius type. The cyclic polarisation (within the −1 V to +1 V range) of the Pt|electrolyte|Pt and Cu|electrolyte|Cu cells shows that the deposition of copper from the electrolyte is basically feasible and reversible. The Cu 2++2e −⇌Cu redox process, which takes place in the presence of the weak ligand CF 3SO − 3, [PAN–PC–Cu(CF 3SO 3) 2 system] is approximately two orders of magnitude slower than the Cu X 2− n n +2e −⇌Cu+ nX − reaction (X=Cl and Br) which occurs in the case of the PAN–PC–CuCl 2 and PAN–PC–CuBr 2 electrolytes.