Counter-diffusion Method Research Articles

A numerical model of protein crystallization with counter diffusion method

A theoretical model is presented to simulate the growth and nucleation of protein lysozyme crystals by counter diffusion method. The comparison of experimental and simulation results shows that the numerical solution accurately describes the initial stages of crystallization. The developed model can be used to describe crystallization processes with various counter diffusion experiment parameters.

Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 A resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

対向拡散法による金属有機構造体膜の開発

対向拡散法による金属有機構造体膜の開発

Crystallization and preliminary X-ray diffraction study of phosphopantetheine adenylyltransferase from M. tuberculosis crystallizing in space group P32

Crystals of M. tuberculosis phosphopantetheine adenylyltransferase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 2.00-A resolution. The crystals belong to sp. gr. P32 and have the following unit-cell parameters: a = b = 106.47 A, c = 71.32 A, α = γ = 90°, β = 120°. The structure was solved by the molecular-replacement method. There are six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is a biologically active form of phosphopantetheine adenylyltransferase from M. tuberculosis.

Structure of recombinant prolidase from Thermococcus sibiricus in space group P21221.

The crystal structure of recombinant prolidase from Thermococcus sibiricus was determined by X-ray diffraction at a resolution of 2.6 Å and was found to contain a tetramer in the asymmetric unit. A protein crystal grown in microgravity using the counter-diffusion method was used for X-ray studies. The crystal belonged to space group P21221, with unit-cell parameters a = 97.60, b = 123.72, c = 136.52 Å, α = β = γ = 90°. The structure was refined to an Rcryst of 22.1% and an Rfree of 29.6%. The structure revealed flexible folding of the N-terminal domain of the protein as well as high variability in the positions of the bound metal ions. The coordinates of the resulting model were deposited in the Protein Data Bank as entry 4rgz.

Effect of Temperature on Synthesis of ZIF-8 Membranes for Propylene/propane Separation by Counter Diffusion Method

Effect of Temperature on Synthesis of ZIF-8 Membranes for Propylene/propane Separation by Counter Diffusion Method

Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 A resolution. The crystals belong to sp. gr. P21 and have the following unit-cell parameters: a = 74.1 A, b = 110.2 A, c = 88.2 A, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

Crystallization and preliminary X-ray diffraction study of porcine carboxypeptidase B

Crystals of porcine pancreatic carboxypeptidase B have been grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction study showed that the crystals belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 79.58 A, c = 100.51 A; α = β = γ = 90.00°. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one of the grown crystals at the SPring 8 synchrotron facility to 0.98 A resolution.

Growth Behavior of Monohydrocalcite (CaCO3·H2O) in Silica-Rich Alkaline Solution

Monohydrocalcite (CaCO3·H2O) has been crystallized in silica-rich alkaline water solution. The crystallization proceeds by a counterdiffusion method without the presence of any other additive except dissolved silica. The crystallization has been followed in situ by optical microscopy and Raman microspectroscopy, while the time evolution of the pH and the concentration of calcium and silica species in solution have also been followed either by in situ (pH) or ex-situ (calcium and silica) time-lapse analysis. The growth of monohydrocalcite particles occurs by different mechanisms that are related to the pH and the rate of pH change with time. The initial peanut-like crystal converts into an onion-like multilayered texture, which is built up by the alternation of loose layers and compact layers as a result of different levels of silica incorporation. A final silica-rich skin covers the hemisphere inhibiting the further growth of monohydrocalcite. When the silica skin fails to cover the whole surface of the hemisphere, a bulge of monohydrocalcite grows from the uncovered area until a new silica skin inhibits its growth except from a small uncovered area from which a new bulge forms. The iteration of this mechanism creates particles with caterpillar-like morphology. Our results show that silica plays a key role in the morphogenesis and texture of monohydrocalcite crystallization due to the coupled interaction between the reverse solubility of silica and carbonate versus pH.

ZIF-8 membranes prepared at miscible and immiscible liquid–liquid interfaces

ZIF-8 membranes are currently attracting attention for the separation of propylene/propane based on the molecular sieve effect. We have recently reported the preparation of ZIF-8 membranes using the counter-diffusion method, which is advantageous in reducing defects in the selective layer. In the present study, ZIF-8 membranes were prepared at the interface of miscible and immiscible pairs of solvents such as water/methanol and water/1-octanol, respectively. ZIF-8 membranes prepared from the immiscible pair of water and 1-octanol showed a propylene permeance of 5.2×10−9molm−2s−1Pa−1 and a propylene/propane permselectivity of 7.2 at 25°C. As for the ZIF-8 membrane prepared by the initial reaction using the immiscible pair of water/1-octanol and the successive reaction using the miscible pair of water/methanol mixture, the average propylene permeance and average propylene/propane permselectivity increased to 1.2×10−8molm−2s−1Pa−1 and 20 at 25°C, respectively. The successive reactions in water/1-octanol and water/methanol mixtures successfully decreased the defects in the ZIF-8 selective layer, resulting in high propylene permeance and high permselectivity for propylene/propane. The structure and gas permeation properties were also thoroughly characterized.

Improving propylene/propane separation performance of Zeolitic-Imidazolate framework ZIF-8 Membranes

Zeolitic-imidazolate framework ZIF-8 has shown great potential for high resolution separation of propylene/propane mixtures based on the size exclusion principle. Recently, we have successfully prepared highly propylene-permselective ZIF-8 membranes by developing a counter diffusion-based in situ method. As a follow-up study, here, we attempted to further improve the separation performance of ZIF-8 membranes prepared by the counter-diffusion method by controlling membrane microstructures via manipulating important synthesis variables as well as activation processes.Important synthesis variables investigated include the sodium formate to ligand ratios, the nature of zinc salts. With a sodium formate to ligand ratio of 0.12 and a mixture of zinc nitrate and zinc chloride, we have successfully synthesized ZIF-8 membranes with much improved propylene/propane separation performances with the separation factor of ~70 and the propylene permeance of ~268×10−10mol/m2sPa. Furthermore, solvothermal activation processes were found to be effective in significantly increasing the propylene permeance while maintaining decent propylene/propane separation factors.

Thickness Reduction of the Zeolitic Imidazolate Framework-8 Membrane by Controlling the Reaction Rate during the Membrane Preparation

The layer thickness of a zeolitic imidazolate framework-8 (ZIF-8) membrane was successfully reduced in order to obtain high permeance of propylene. The ZIF-8 membranes for propylene/propane separation were prepared using a counter-diffusion method by changing the solution concentration and the molar ratio of 2-methylimidazole to zinc ion (Hmim/Zn2+). ZIF-8 layers with different thicknesses were formed in the outermost regions of porous α-alumina capillary substrates, and the minimum thickness of the ZIF-8 layer was 5 µm. Single-component gas permeation properties were measured using propylene and propane at a temperature of 298 K, and the permeability, diffusivity, and solubility were analyzed. The maximum propylene/propane permselectivity of 135, diffusivity selectivity of 125, and solubility selectivity of 1.1 were obtained at a Hmim/Zn2+ ratio of 1 with a propylene permeance of 1.1×10−9 mol·m−2·s−1·Pa−1. By contrast, a propylene/propane permselectivity of 113 with a propylene permeance of 3.3×10−9 mol·m−2·s−1·Pa−1 was obtained at a Hmim/Zn2+ ratio of 0.33. The thickness of the ZIF-8 layer, the permselectivity of propylene/propane, and the permeance of propylene were controlled by regulating the concentration and the Hmim/Zn2+ ratio for the synthesis of the ZIF-8 membrane by the counter-diffusion method.

Crystallographic Analysis of Ground and Space Thermostable T1 Lipase Crystal Obtained via Counter Diffusion Method Approach

Three-dimensional structure of thermostable lipase is much sought after nowadays as it is important for industrial application mainly found in the food, detergent, and pharmaceutical sectors. Crystallization utilizing the counter diffusion method in space was performed with the aim to obtain high resolution diffracting crystals with better internal order to improve the accuracy of the structure. Thermostable T1 lipase enzyme has been crystallized in laboratory on earth and also under microgravity condition aboard Progress spacecraft to the ISS in collaboration with JAXA (Japanese Aerospace Exploration Agency). This study is conducted with the aims of improving crystal packing and structure resolution. The diffraction data set for ground grown crystal was collected to 1.3 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.40 Å, b = 80.95 Å, and c = 99.81 Å, whereas the diffraction data set for space grown crystal was collected to 1.1 Å resolution and belonged to monoclinic C2 space group with unit cell parameters a = 117.31 Å, b = 80.85 Å, and c = 99.81 Å. The major difference between the two crystal growth systems is the lack of convection and sedimentation in microgravity environment resulted in the growth of much higher quality crystals of T1 lipase.

Purification, crystallization and preliminary crystallographic analysis of the ligand-binding regions of the PctA and PctB chemoreceptors from Pseudomonas aeruginosa in complex with amino acids.

Pseudomonas aeruginosa is an opportunistic pathogen and one of the major model organisms for the study of chemotaxis. The bacterium harbours 26 genes encoding chemoreceptors, most of which have not been annotated with a function. The paralogous chemoreceptors PctA and PctB (Pseudomonas chemotactic transducer A and B) were found to mediate chemotaxis towards L-amino acids. However, the ligand spectrum of the receptors is quite different since the recombinant ligand-binding region (LBR) of PctA binds 17 different L-amino acids whereas that of PctB recognizes only five. To determine the molecular basis underlying this ligand specificity, PctA-LBR and PctB-LBR have been purified and crystals have been produced after pre-incubation with L-Ile and L-Arg, respectively. Initial crystallization conditions have been identified by the counter-diffusion method and X-ray data have been collected at 2.5 Å (PctA-LBR bound to L-Ile) and 3.14 Å (PctB-LBR bound to L-Arg) resolution. Crystals belonged to space groups P2(1)2(1)2(1) and P3(1)2(1), with unit-cell parameters a = 72.2, b = 78.5, c = 116.6 Å and a = b = 111.6, c = 117.4, respectively, for PctA-LBR and PctB-LBR. Molecular-replacement methods will be pursued for structural determination.

Diffusive separation of propylene/propane with ZIF-8 membranes

The contribution of diffusive separation of propylene/propane was determined from the permeation properties of zeolitic-imidazolate framework-8 (ZIF-8)11ZIF, zeolitic-imidazolate framework. membranes. These membranes were prepared using a counter diffusion method, resulting in the formation of an 80μm-thick ZIF-8 layer on the outermost section of a porous α-alumina capillary substrate. Single-component gas permeation properties were investigated using helium, hydrogen, carbon dioxide, oxygen, nitrogen, methane, propylene, and propane at temperatures ranging from 298 to 363K. The molecular sieve gas permeation properties of the ZIF-8 membranes increased with reaction time, which is consistent with the evolution of ZIF-8 crystal formation. The ZIF-8 membrane permeances of hydrogen and propylene reached 9.1×10−8 and 2.5×10−9molm−2s−1Pa−1, respectively. The ideal separation factors for hydrogen/propane and propylene/propane at 298K were found to be 2000 and 59, respectively. The diffusion separation factor increased with decreasing temperature, reaching 23, while the solution separation factor remained 2.7. Therefore, the separation of propylene/propane was attributed to be mainly governed by diffusive separation.

Liesegang-like patterns of Toll crystals grown in gel

Generating high-quality crystals remains a bottleneck in biological and materials sciences. Here a counter-diffusion method was used to improve the X-ray diffraction quality of the N-terminal domain of Drosophila melanogaster Toll receptor crystals. It was observed that crystallization occurred with a peculiar pattern along the capillary resembling Liesegang bands; this phenomenon is described at both macroscopic and atomic levels. It was found that bands appeared for native protein as well as for co-crystals of magic triangle (I3C)-bound protein even though they crystallize in different space groups. Crystallization occurred with a linear recurrence independent of the precipitant concentration and a protein-specific spacing coefficient. Bandwidth varied along the capillary, oscillating between large precipitation areas and single crystals. The reported data suggest that repetitive patterns can be generated with biological macromolecules in the presence of sodium malonate as a crystallization agent. A comparison with typical Liesegang patterns and the possible mechanism underlying this phenomenon are discussed.

Crystallization of monohydrocalcite in a silica-rich alkaline solution

Monohydrocalcite was crystallized in a silica-rich alkaline solution at room temperature using the counter diffusion method (CDM) in the absence of magnesium, by the reaction of calcium chloride and sodium carbonate. Field emission scanning electron microscopy observations showed monohydrocalcite crystals that were hundreds of micrometers in size exhibiting a unique multi-layered structure. X-ray diffraction and Raman microspectroscopy characterizations demonstrated that monohydrocalcite remained in a stable phase for a number of months. After monitoring the crystallization process by in situ Raman microspectroscopy, it was found that monohydrocalcite was the initial phase, with no phase transformation occurring during the crystal growth. This work demonstrates that silica plays a key role in the formation and stabilization of the monohydrocalcite phase.

Metal–organic framework membranes with layered structure prepared within the porous support

A uniquely microstructured membrane, a metal–organic framework (MOF) layer within the porous support, was prepared by a counter-diffusion method. The MOF-based membrane prepared from the copper benzene tricarboxylate (Cu-BTC), one of the MOFs, exhibited excellent gas perm-separation properties with an ideal H2/CH4 selectivity of 153.

X-ray study of the conformational changes in the molecule of phosphopantetheine adenylyltransferase fromMycobacterium tuberculosisduring the catalyzed reaction

Structures of recombinant phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis (PPATMt) in the apo form and in complex with the substrate ATP were determined at 1.62 and 1.70 Å resolution, respectively, using crystals grown in microgravity by the counter-diffusion method. The ATP molecule of the PPATMt-ATP complex was located with full occupancy in the active-site cavity. Comparison of the solved structures with previously determined structures of PPATMt complexed with the reaction product dephosphocoenzyme A (dPCoA) and the feedback inhibitor coenzyme A (CoA) was performed using superposition on C(α) atoms. The peculiarities of the arrangement of the ligands in the active-site cavity of PPATMt are described. The conformational states of the PPAT molecule in the consequent steps of the catalyzed reaction in the apo enzyme and the enzyme-substrate and enzyme-product complexes are characterized. It is shown that the binding of ATP and dPCoA induces the rearrangement of a short part of the polypeptide chain restricting the active-site cavity in the subunits of the hexameric enzyme molecule. The changes in the quaternary structure caused by this rearrangement are accompanied by a variation of the size of the inner water-filled channel which crosses the PPAT molecule along the threefold axis of the hexamer. The molecular mechanism of the observed changes is described.

Crystallization of uridine phosphorylase from Shewanella oneidensis MR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis.

Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2(1) and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data.