Chiral crystallization of glutamic acid on self assembled films of cysteine

In this article, we describe the preparation and use of chiral surfaces derived from enantiomerically pure crystals of amino acids. For this purpose, we chose to employ a self-assembly process to grow nanoscale chiral films of (+)-L or (-)-D cysteine, onto gold surfaces. We utilized those chiral films as resolving auxiliaries in the crystallization of enantiomers from solutions. To demonstrate the chiral discriminating ability of the chiral surfaces in crystallization processes, we investigated the crystallization of rac-glutamic acid onto the chiral films. Our study demonstrates the potential application of chiral films to control chirality throughout crystallization, where one enantiomer crystallizes on the chiral surfaces with relatively high enantiomeric excess. In addition, crystallization of pure glutamic acid enantiomers, and its racemic compound on to chiral films resulted in crystal morphology modification with preferred crystal orientation, which assists in the interpretation of the ability of our chiral surfaces to function as chiral selectors.     

离子液体1-丁基-3-甲基咪唑氯代盐对溶菌酶结晶的影响

以亲水性离子液体1-丁基-3-甲基咪唑氯盐(BmimCl)为添加剂,研究离子液体对溶菌酶结晶的影响.分别考察了离子液体对溶菌酶晶体数量与尺寸、晶体形貌及蛋白质纯度的影响,并探讨了离子液体对结晶过程影响的作用机制.离子液体通过增大溶菌酶的溶解度和其自身低蒸气压两种途径,降低了溶菌酶在结晶过程中的过饱和度,更有利于晶体的成核和生长,得到更好的结果.如避免多晶态现象的发生,增大晶体的尺寸,降低溶菌酶样品纯度的要求.X-射线衍射分析表明,离子液体未改变晶体的晶型结构,但可提高晶体的衍射分辨率.     

Comparison of two different lysozyme types under native and crystallization conditions using two-dimensional NMR and dynamic light scattering

In order to elucidate differences observed in the aggregation kinetics of hen-egg white lysozyme under crystallization conditions we have undertaken a comparative study of the enzyme marketed by Seikagaku and Sigma companies. When the crystallization of the two lysozyme preparations is followed by time-resolved dynamic light scattering, the structural differences are also observed under native conditions in the early nucleation kinetics. The differences are manifested in the formation rates of macroscopic crystals, but do not influence the morphology of the typical tetragonal lysozyme crystal. Using two-dimensional NMR we have followed the differences in the native-like solution structure of the two preparations, while the primary sequence and molecular mass are identical. According to the published structure of tetragonal lysozyme crystal the largest deviations were found for the residues involved in the intermolecular interactions in crystal structure.     

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.     

Comparative crystallization behavior of polymers and aqueous solutions.

This paper gives a short introduction to the modes of crystallization of polymers and shows, by a series of micrographs, that the resulting morphology is very similar to that of ice crystals growing from aqueous solutions. The similarity is explained by similar conditions of nucleation and growth, leading in both cases to a hindered and partial crystallization. It is shown that the resulting crystal patterns can be qualitatively explained by estimating the relative growth rates in different directions on a developing crystal face as a function of supercooling and concentration.     

Parameters for crystal growth of ribosomal subunits

A systematic analysis of the parameters that control the crystal growth of the large subunit of ribosomes from B stearothermophilus has been carried out. Several parameters have been identified and classified according to their significance. It was found that only biologically active particles can crystallize and that the critical period for the crystallization process is the first few days, during which changes in the volume and content of the crystallization drop are of importance for both nucleation and crystal growth. Consequently, an experimental procedure for fine control of these variables has been developed. As a result of these studies, the reproducibility of crystal formation was increased, and larger and more stable crystals have been obtained.     

A simple apparatus for controlling nucleation and size in protein crystal growth

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the "hanging drop" method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.     

Trypsin crystallization by membrane-based techniques

To grow protein crystals is not an easy task; moreover, if we need to grow protein crystals with controlled shape, size, and size distribution, depending on their application, the mission becomes even harder. Membrane crystallization has been recognized as an interesting tool for growing protein crystals with enhanced crystallization kinetics, both in static and in forced solution flow configuration, without detrimental effects on crystal quality. In the present work, we have studied the membrane crystallization process of benzamidine inhibited trypsin from bovine pancreas (BPT), with ammonium sulphate (dissolved in Tris-HCl buffer, 0.1 M, pH 8.5), as precipitant agent. We have demonstrated that, by using the membrane crystallization technique, BPT crystals can be obtained in 24-48 h, in static configuration, and in 4-7 days, in a forced solution flow system, depending on the experimental conditions. Furthermore, the kinetics of BPT crystallization have been modulated, to control the morphological characteristics of the crystals produced, by an accurate selection of the operative parameters involved in the process. The active membrane surface and the flow rate of extraction solvent in quiescent configuration, and the solution velocity in forced convection solution experiments, were the parameters investigated. In this respect, membrane crystallization techniques have been assessed as an interesting way for growing proteins, and more specifically enzyme crystals, with high control on the final properties of the crystalline material produced, with potential fundamental implication in the field of structural biology and biotechnology.     

Drop-on-Demand System for Manufacturing of Melt-based Solid Oral Dosage: Effect of Critical Process Parameters on Product Quality

The features of a drop-on-demand-based system developed for the manufacture of melt-based pharmaceuticals have been previously reported. In this paper, a supervisory control system, which is designed to ensure reproducible production of high quality of melt-based solid oral dosages, is presented. This control system enables the production of individual dosage forms with the desired critical quality attributes: amount of active ingredient and drug morphology by monitoring and controlling critical process parameters, such as drop size and product and process temperatures. The effects of these process parameters on the final product quality are investigated, and the properties of the produced dosage forms characterized using various techniques, such as Raman spectroscopy, optical microscopy, and dissolution testing. A crystallization temperature control strategy, including controlled temperature cycles, is presented to tailor the crystallization behavior of drug deposits and to achieve consistent drug morphology. This control strategy can be used to achieve the desired bioavailability of the drug by mitigating variations in the dissolution profiles. The supervisor control strategy enables the application of the drop-on-demand system to the production of individualized dosage required for personalized drug regimens.     

SERS and Antibacterial Active Green Synthesized Gold Nanoparticles

Accurate control of size, composition, morphology, and stability, and the use of environmentally friendly procedures are highly desirable for the synthesis of nanoparticles. Here is a report on the use of Vitis californica leaf broth for the synthesis of gold nanoparticles. The morphology of the particles formed consists of a mixture of gold nanotriangles and spheres with fcc (111) structure. At lower concentrations of the extract, formation of triangular-shaped particles is found to dominate, while at higher concentrations, almost spherical particles alone are observed. The investigations made on the surface enhanced Raman scattering activity of these nanoparticles using 2-aminothiophenol and crystal violet as probe molecules are discussed in detail. The synthesized nanoparticles displayed efficient antibacterial activity against the tested gram-negative bacterium Escherichia coli and gram-positive bacterium Staphylococcus aureus.     

Influence of divalent cations in protein crystallization.

We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.     

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.     

The effect of protein impurities on lysozyme crystal growth

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ?110? and ?101? lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.     

Calculations on crystal packing of a flexible molecule, Leu-enkephalin

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.     

Nanogold hollow balls with dendritic surface for hybridization of DNA

Au hollow balls are fabricated by adsorption of gold 3.5 nm in diameter onto a mixed vesicle composed of mixed polymerized diacetylene which made of negative charged 10,12-pentacosadiynoic acid (PCDA) and positive charged 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH(2)). The morphology of these hollow spheres could be controlled by changing the ratio of PCDA and PCDA-NH(2) and the immobilization and hybridization ability of the gold hollow ball have been investigated using a quartz crystal microbalance (QCM). It was found that a dendritic surface in an appropriate ratio existed. The hybridization amount of target DNA is about three to five times for the Au-mixed hollow ball at an optimal ratio (PCDA/PCDA-NH(2)=1/3) as compared with that for pure Au-PCDA-NH(2), though the immobilization amount of ssDNA on these two samples are almost the same, and the detected limitation of target DNA is extend from 10(-9) to 10(-12) M. The stability against breakage by transportation, combined with the simplicity and efficiency of detection, would offer an important advantage over unpolymerized one. This result shows the possibility to control the morphology and surface of nanogold hollow spheres by changing the ratio of PCDA and PCDA-NH(2) for the develop of a better DNA detection assay, further proving the idea that low surface coverage and higher DNA probe to target DNA ratios lead to optimal hybridization.     

Inhibition of protein crystallization by evolutionary negative design

Why are proteins so hard to crystallize? We propose an 'evolutionary negative design' principle to explain this difficulty. Proteins have evolved to avoid crystallization because crystallization compromises the viability of the cell. Evolutionary negative design is supported by much evidence in the literature, including the effect of mutations on the crystallizability of a protein, the correlations found in the properties of crystal contacts in bioinformatics databases, and the positive use of protein crystallization by bacteria and viruses.     

Study of the synthesis, crystallization, and morphology of poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers

Poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers PEG-PCL were synthesized by ring-opening polymerization of epsilon-caprolactone using monomethoxy poly(ethylene glycol) as the macroinitiator and calcium ammoniate as the catalyst. Obvious mutual influence between PEG and PCL crystallization was studied by altering the relative block length. Fixing the length of the PEG block (Mn = 5000) and increasing the length of the PCL block, the crystallization temperature of the PCL block rose gradually from 1 to about 35 degrees C while that of the PEG block dropped from 36 to -6.6 degrees C. Meanwhile, the melting temperature of the PCL block went up from 30 to 60 degrees C, while that of the PEG block declined from 60 to 41 degrees C. If the PCL block was longer than the PEG block, the former would crystallize first when cooling from a molten state and led to obviously imperfect crystallization of PEG and vice versa. And they both crystallized at the same temperature, if their weight fractions were equal. We found that the PEG block could still crystallize at -6.6 degrees C even when its weight fraction is only 14%. A unique morphology of concentric spherulites was observed for PEG5000-PCL5000. According to their morphology and real-time growth rates, it is concluded that the central and outer sections in the concentric spherulites were PCL and PEG, respectively, and during the formation of the concentric spherulite, the PEG crystallized quickly from the free space of the PCL crystal at the earlier stage, followed by outgrowing from the PCL spherulites in the direction of right angles to the circle boundaries until the entire area was occupied.     

Self-assembly of precursors in single-crystal growth of biopolymers

Self-assembly of precursors in dilute solution single-crystal growth of poly[(R)-3-hydroxyvalerate] (PHV) and the fungal polysaccharide mycodextran were studied by transmission electron microscopy, especially at the early stages of crystallization. Precursors for PHV, such as small primary nuclei and tiny square tiles, consolidate to a large square crystal composed of orthogonally arranged tiles. By contrast, the precursors of mycodextran were lath-shaped, which suggests that the crystal growth is mainly in the longitudinal direction. Needlelike precursors were observed either free or as protrusions at the lath ends. In general, single-crystal shape and morphology are dependent on the type of precursor and on the manner of crystallization.     

A new generation of sodium chloride porogen for tissue engineering

Porogen leaching is a widely used and simple technique for the creation of porous scaffolds in tissue engineering. Sodium chloride (NaCl) is the most commonly used porogen, but the current grinding and sieving methods generate salt particles with huge size variations and cannot generate porogens in the submicron size range. We have developed a facile method based on the principles of crystallization to precisely control salt crystal sizes down to a few microns within a narrow size distribution. The resulting NaCl crystal size could be controlled through the solution concentration, crystallization temperature, and crystallization time. A reduction in solution temperature, longer crystallization times, and an increase in salt concentration resulted in an increase in NaCl crystal sizes due to the lowered solubility of the salt solution. The nucleation and crystallization technique provides superior control over the resulting NaCl size distribution (13.78 ± 1.18 μm), whereas the traditional grinding and sieving methods produced NaCl porogens 13.89 ± 12.49 μm in size. The resulting NaCl porogens were used to fabricate scaffolds with increased interconnectivity, porous microchanneled scaffolds, and multiphasic vascular grafts. This new generation of salt porogen provides great freedom in designing versatile scaffolds for various tissue-engineering applications.