Counter-diffusion Method Research Articles

Perfect Crystals: microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography

The NASA mission Perfect Crystals used the microgravity environment on the International Space Station (ISS) to grow crystals of human manganese superoxide dismutase (MnSOD)—an oxidoreductase critical for mitochondrial vitality and human health. The mission’s overarching aim is to perform neutron protein crystallography (NPC) on MnSOD to directly visualize proton positions and derive a chemical understanding of the concerted proton electron transfers performed by the enzyme. Large crystals that are perfect enough to diffract neutrons to sufficient resolution are essential for NPC. This combination, large and perfect, is hard to achieve on Earth due to gravity-induced convective mixing. Capillary counterdiffusion methods were developed that provided a gradient of conditions for crystal growth along with a built-in time delay that prevented premature crystallization before stowage on the ISS. Here, we report a highly successful and versatile crystallization system to grow a plethora of crystals for high-resolution NPC.

Robust ZIF-8 and its derivative composite membrane for antibiotic desalination with high performance

Zeolitic imidazolate framework (ZIF) membranes have great potential applications in nanofiltration. However, preparing continuous and intact ZIF membranes on the surface of a substrate remains challenging. In this study, we assembled ZIF-8 nanoparticles in situ in the pores of a porous alumina substrate via a freezing-assisted counter-diffusion method. Although a continuous ZIF-8 membrane was not formed on the surface of the substrate, the obtained nano-confined composite membrane was dense enough to separate antibiotics and salt. The rejection rate of tetracycline was above 99 %, while that of sodium chloride was below 20 %, demonstrating good antibiotic desalination performance. The permeance of the ZIF-8 membrane reached 23.5 LMH/bar. Due to the nano-confined structure, the obtained ZIF-8 composite membrane was robust. Besides, polishing the surface layer did not reduce the separation performance. Moreover, sulfurizing the ZIF-8 membrane improved its stability in water. It also improved the permeance to 31.3 LMH/bar, which remained stable for 100 h. Therefore, the obtained robust ZIF-8 membrane and its derivative composite membrane fabricated via freezing-assisted counter-diffusion have great potential in antibiotic desalination.

2D nanosheets seeding layer modulated covalent organic framework membranes for efficient desalination

Covalent organic frameworks (COFs) are emergent ideal membrane materials, owing to their pre-designable and well-defined pore structures. However, COFs typically exist as solid powder and are hard to dissolve in most solvent, which renders COFs poor processability in membrane formation. In this study, TpPa-SO3H/PAN COF membranes (COFMs) were prepared via counter diffusion method, which is modulated by the pre-assembled two-dimensional (2D) crystalline TpPa-SO3H nanosheets as seeding layer. The TpPa-SO3H seeding layer regulated the diffusion-reaction behavior of monomers and induced the confined growth of TpPa-SO3H COFs, which is favorable for eliminating the intergranular defects inside the TpPa-SO3H/PAN COFMs. The thickness of TpPa-SO3H seeding layer can be controlled by the amount of TpPa-SO3H nanosheets. The charge of the channel wall inside the membrane can fortify the electrostatic repulsion mechanism and facilitate efficient desalination. Under the optimal conditions, the Na2SO4 rejection rate of TpPa-SO3H/PAN COFMs can reach 97.4%. Unlike conventional polyamide membranes whose desalination performance usually decreases with the increase of salt concentration, the TpPa-SO3H/PAN COFMs preserved superior desalination performance toward high salt concentration up to 10,000 ppm.

Crystallization in Microgravity and the Atomic-Resolution Structure of Uridine Phosphorylase from Vibrio cholerae

Uridine phosphorylases are known as key targets for the development of new anticancer and antiparasitic agents. Crystals of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae were grown in microgravity by the capillary counter-diffusion method on board of the International Space Station. The three-dimensional structure of this enzyme was determined at atomic (1.04 Å) resolution (RCSB PDB ID: 6Z9Z). Alternative conformations of long fragments (β-strands and adjacent loops) of the protein molecule were found for the first time in the three-dimensional structure of uridine phosphorylase in the absence of specific bound ligands. Apparently, these alternative conformations are related to the enzyme function. Conformational analysis with Markov state models demonstrated that conformational rearrangements can occur in the ligand-free state of the enzyme.

Preparation, Crystallization, and Preliminary X-Ray Diffraction Study of Mutant Carboxypeptidase T Bearing the Primary Specificity Pocket and the Active-Site Loop of Carboxypeptidase B

Crystals of mutant carboxypeptidase T from Thermoactinomyces vulgaris (CPT11QG) with amino-acid substitutions G215S, Q249G, A251G, T257A, D260G, T262D, and L254I and with the insertion ins253T were grown in microgravity by the capillary counter-diffusion method. The crystals belong to sp. gr. P31, which differs from the space group of the wild-type enzyme (P6322). The X-ray diffraction data set was collected from the crystals at the SPring-8 synchrotron facility (Japan) and is suitable for crystal structure determination at 2.45 A resolution.

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

Comparison of transient and pseudo-steady diffusion of methane in coal and implications for coalbed methane control

Diffusion coefficient is a key parameter characterizing the methane diffusion capacity. Because of the differences between transient diffusion and pseudo-steady diffusion in methane diffusion rate, the diffusion processes and diffusion coefficients are different. In this study, the transient diffusion and pseudo-steady diffusion properties were measured via particle method and counter diffusion method, respectively. The results indicated that the pseudo-steady diffusion coefficient is higher than the transient diffusion coefficient, and the difference may be attributed to the influence of surface diffusion of adsorbed methane. The matrix shape factor is also a key parameter influencing the methane diffusivity, and it has a higher influence on diffusion amount of methane than the diffusion coefficient. It is suggested that for methane extraction project, the pseudo-steady diffusion coefficient and coal matrix shape factor should be considered, while transient diffusion coefficient should be used to calculate the indices for evaluating the risk of coal and gas outburst.

Electrokinetic control of the growth of gypsum aggregates in compacted montmorillonite

Mineral precipitation and its impact on the barrier functions of clayey materials have attracted attention in the geological disposal of radioactive wastes. In this study, the growth of gypsum aggregates in compacted montmorillonite was investigated using the electrokinetic method. The results demonstrated that the morphology of aggregates can be partially controlled by adjusting the initial concentration of Ca in Na/Ca mixed montmorillonite. In particular, a lower initial Ca concentration maintained for long periods of time engendered the growth of gypsum as a layer of large aggregates up to 700 μm. The layer of gypsum aggregates was made for a short duration (1 day) without excess salt remaining around the layer. The short duration and the absence of excess salt represent the main advantage of the electrokinetic method compared to a counter diffusion method to form artificial precipitates in clayey materials. Techniques to enhance and control aggregates are useful to prepare altered bentonite, which can be used to evaluate the impact of precipitates and their morphologies on the diffusivity and permeability of compacted bentonite.

Changes of Thermostability, Organic Solvent, and pH Stability in Geobacillus zalihae HT1 and Its Mutant by Calcium Ion.

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 °C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability.

Preliminary X-ray Diffraction Study of Macrophage Migration Inhibitory Factor at Near-Atomic Resolution

Crystals of human macrophage migration inhibitory factor in complexes with phenyl isothiocyanate and tartrate were grown by the capillary counter-diffusion method under microgravity conditions. The preliminary X-ray diffraction study of these complexes was performed at near-atomic resolution (1.16 A).

Controlled Self-Assembly and Tuning of Large PbS Nanoparticle Supercrystals

Self-assembly of colloidal nanocrystals (NCs) into ordered superlattices (SLs) and supercrystals (SCs) enables new artificial NC solids for nanoelectronic and nanophotonic applications, which requires critical control of nucleation and growth conditions. Herein large SCs of PbS NCs up to ∼100 μm size were synthesized by two controlled self-assembly methods from NC solutions. Both translational symmetry and orientational ordering of the nanocrystals in the SCs were readily tuned by excess oleic acid ligands and antisolvents. Slow evaporation and the counterdiffusion method of solvents resulted in the formation of single SCs with two different SLs from the same PbS NCs: a face-centered cubic SL with weak yet complex orientational order or a body-centered cubic SL with strong and uniform particle orientation, respectively. The translational ordering was mainly determined by the effective shape of the NCs while the difference in orientational order was a result of the balance between ligand–ligand attraction and rotational entropy. The ease of the growth of large SC solids could lead to diverse NC systems and facilitate essential investigation of nanoparticle interactions and coupling based nanoelectronic and nanophotonic properties.

Crystallization and Preliminary X-ray Diffraction Study of Purine Nucleoside Phosphorylase from the Thermophilic Bacterium Thermus thermophilus Strain HB27

Recombinant purine nucleoside phosphorylase from the thermophilic Thermus thermophilus strain encoded by the TT_C0194 gene was purified to homogeneity. The crystallization conditions for the enzyme were found by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown under microgravity conditions by the capillary counter-diffusion method. The crystals belong to sp. gr. P212121 and have the following unit-cell parameters: a = 89.9 A, b = 121.0 A, c = 215.7 A, α = β = γ = 90°. The X-ray diffraction data set suitable for the determination of the three-dimensional structure of purine nucleoside phosphorylase was collected from the grown crystals at the SPring-8 synchrotron facility to 2.5 A resolution.

Preparation of zeolite imidazolate framework/graphene hybrid aerogels and their application as highly efficient adsorbent

Zeolite imidazolate framework/graphene aerogels (ZIF/GAs) were synthesized via a counter diffusion method for wastewater purification. XRD, FTIR, Raman, XPS spectroscopy, and FESEM technique were applied to characterize the structure and morphology of the aerogels, and the results confirmed that the ZIF/GAs exhibited a 3D interconnected macroporous framework of graphene sheets with uniform dispersion of ZIF particles. The adsorption performance of the as-prepared ZIF-67/GA and ZIF-8/GA was investigated by adsorbing dyes. The adsorption capacities showed that the ZIF-67/GA had higher adsorption efficiency than the ZIF-8/GA and pristine graphene aerogels towards dyes. The adsorption ability of the ZIF/GAs towards anionic MO dye was relatively larger than that of cationic RhB dye, ascribed to the different electrostatic attraction and π-π interaction of ZIF/GAs to dyes. The adsorption kinetics of the ZIF/GAs towards dyes were studied as well. As such, the prepared ZIF/GAs composites are the perfect candidates for pollutants removal and water purification.

Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure

Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

Crystallization and preliminary X-ray diffraction study of recombinant adenine phosphoribosyltransferase from the thermophilic bacterium Thermus thermophilus strain HB27

Adenine phosphoribosyltransferase (APRT) belongs to the type I phosphoribosyltransferase family and catalyzes the formation of adenosine monophosphate via transfer of the 5-phosphoribosyl group from phosphoribosyl pyrophosphate to the nitrogen atom N9 of the adenine base. Proteins of this family are involved in a salvage pathway of nucleotide synthesis, thus providing purine base utilization and maintaining the optimal level of purine bases in the body. Adenine phosphoribosyltransferase from the extremely thermophilic Thermus thermophilus strain HB27 was produced using a highly efficient E. coli producer strain and was then purified by affinity and gel-filtration chromatography. This enzyme was successfully employed as a catalyst for the cascade biosynthesis of biologically important nucleotides. The screening of crystallization conditions for recombinant APRT from T. thermophilus HB27 was performed in order to determine the enzyme structure by X-ray diffraction. The crystallization conditions, which were found by the vapor-diffusion technique, were then optimized to apply the counter-diffusion technique. The crystals of the enzyme were grown by the capillary counter-diffusion method. The crystals belong to sp. gr. P1211 and have the following unitcell parameters: a = 69.86 A, b = 82.16 A, c = 91.39 A, α = γ = 90°, β = 102.58°. The X-ray diffraction data set suitable for the determination of the APRT structure at 2.6 A resolution was collected from the crystals at the SPring-8 synchrotron facility (Japan).

Formation of ZIF-8 membranes inside porous supports for improving both their H2/CO2 separation performance and thermal/mechanical stability

Zeolitic imidazolate framework-8 (ZIF-8) membranes are highly suitable as H2-selective walls in membranes reactors used for water gas shift reactions because of their efficient molecular sieving properties that favor H2 transport, coupled with their high thermal stability and processability. In this study, we adopted an in-situ counter diffusion method to fabricate a ZIF-8 membrane; Zn sources, already placed inside a porous support, were allowed to diffuse out and react with the 2-methylimidazole (mim) molecules in the bulk phase. Because the reaction rates between the Zn source and the mim molecule were very high, their diffusion rates played a key role in determining the final properties of the membranes. To control the diffusion rate, a hierarchically structured support, i.e., a γ-Al2O3 layer-coated α-Al2O3 disc (γ-/α-Al2O3 disc), was used in addition to an intact α-Al2O3 disc. ZIF-8 membranes in the α-Al2O3 disc (membrane ZIF-8_α) were primarily formed on top similar to a conventional supported-membrane, whereas those in the γ-/α-Al2O3 disc (membrane ZIF-8_γα) were produced inside the support. As desired, membrane ZIF-8_γα showed marked H2 separation performance with a maximum (max) H2/CO2 separation factor (SF) of ~9.9 ± 1.2 at 250°C (vs. a max H2/CO2 SF of ~7.5 ± 0.2 for membrane ZIF-8_α). Although both type membranes persisted at 200 and 250°C for up to 72h, at a higher temperature of 300°C, the membrane performance started deteriorating after ~2h and ~10h for membranes ZIF-8_α and ZIF-8_γα, respectively. This indicates that the γ-Al2O3 layer served as a protective layer for preserving the performance of the ZIF-8 membrane. The performance at 300°C was completely degraded due to the eventual conversion of ZIF-8 into ZnO phases.

Three-dimensional structure of porcine pancreatic carboxypeptidase B with an acetate ion and two zinc atoms in the active site

Crystals of porcine pancreatic carboxypeptidase B (CPB) were grown by the capillary counter-diffusion method in the presence of polyethylene glycol and zinc acetate. The three-dimensional structure of CPB was determined at 1.40 A resolution using the X-ray diffraction data set collected from the crystals of the enzyme at the SPring 8 synchrotron facility and was refined to Rfact = 17.19%, Rfree = 19.78%. The structure contains five zinc atoms, two of which are present in the active site of the enzyme, and an acetate ion. The arrangement of an additional zinc atom in the active site and the acetate ion is different from that reported by Yoshimoto et al.

Apparent and True Diffusion Coefficients of Methane in Coal and Their Relationships with Methane Desorption Capacity

The diffusion coefficient of methane in coal is a key parameter for the prediction of coalbed methane production. The apparent diffusion coefficient is different from the true diffusion coefficient, which would result in the deviation of methane production. In this study, the particle method using the unipore model and the counterdiffusion method is adopted to measure the methane diffusion coefficients. The results indicated that the true diffusion coefficient obtained by the counterdiffusion experiment decreases first and then increases with increasing methane pressure. The apparent diffusion coefficients obtained by the particle method with two different grain sizes are lower than the true diffusion coefficient. The relationship between the apparent diffusion coefficient and the true diffusion coefficient is analyzed, and the desorption capacity factor (DCF) is proposed to reflect the gap between them. The apparent diffusion coefficient is closer to the true diffusion coefficient when the DCF is small. ...

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°С, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-A resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 A, b = 112.6 A, c = 110.2 A, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-A resolution.

The effect of glycine on the growth of calcium carbonate in alkaline silica gel

Calcium carbonate was crystallized in alkaline silica gel with the presence of glycine. The crystallization proceeded with a counterdiffusion method by the reaction of calcium chloride and sodium carbonate. Optical microscopy observation showed a significant effect of glycine on the morphology control of calcite crystals. When the initial concentration of glycine was high enough (10mg/mL, 20mg/mL), spherical vaterite particles formed in alkaline silica gel concomitantly together with dumbbell shaped calcite particles. The in situ study by micro-Raman spectroscopy demonstrated that both vaterite and the concomitant calcite were stable phases during their growth processes since the initial appearance. A possible mechanism has been discussed to emphasize the effect of glycine on the nucleation of vaterite and the morphological control of calcite.