Deuterated hen egg-white lysozyme crystals: Optimization of the growth conditions and morphology

Deuterated and protonated tetragonal lysozyme crystals are grown using the hanging-drop vapor-diffusion method. The size of the lysozyme crystals grown is determined as a function of the concentration of sodium chloride used as a precipitant. It is found that crystallization leads to the formation of lysozyme crystals with three different habits. Morphological and X-ray diffraction analyses of the deuterated and protonated lysozyme crystals demonstrate that, despite the different habits, all the crystals grown belong to the tetragonal crystal system. The simple forms of lysozyme crystals are revealed. It is shown that the habits of the lysozyme crystals are determined by the specific combinations of simple forms. The mechanisms responsible for the formation of lysozyme crystals with different habits are discussed.     

Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue

The reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach‐Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Crystallization and stability of different protein crystal modifications: A case study of lysozyme

The crystallization, including both the phase diagram and the phase transition of hen egg white lysozyme (HEWL) was investigated. A tetragonal modification and a needle modification were obtained during crystallization. The phase diagram and stability of two modifications in both acid and basic pH solutions (pH 4.5, 8.0 and 9.0) were determined. Besides in acid solutions, the well‐known tetragonal crystals can also be obtained in basic solutions at low temperature (7 °C) while the needle like modification can only crystallize in a basic solution. Based on the phase diagram, phase transfer behavior was found to exist between the two modifications. In basic solutions, tetragonal modification can transfer to needle shaped crystals. This process can be affected by a changing of pH and temperature. While in acid buffer, the needle shaped crystals dissolve and tetragonal crystals crystallize and remain in solution.     

Direct AFM observations of impurity effects on a lysozyme crystal

Impurity effects on the growth of tetragonal lysozyme crystals have been studied using in situ atomic force microscopy. Commercially available hen egg white lysozyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining, and purified by re-crystallization and successive high pressure liquid chromatography. On the (1 1 0) crystal surface, there was no significant difference in morphology between crystals grown in commercial and in purified solutions. On the (1 0 1) surface, however, a large number of small particles were found when the crystal was grown in the commercial solution, while the surface grown in the purified solution was quite smooth. Among the typical residual impurities contained in commercial lysozyme, only covalently bound lysozyme dimer yielded such particles. From measurements of particle separation and an estimate of the critical nucleation size, we infer that the particles reduced the step velocity according to the mechanism described by Cabrera et al. [N. Cabrera, D.A. Vermilyea, in: R.H. Doremus et al. (Eds.), Growth and Perfection of Crystals, 1958, P. 393].     

In situ study of the growth and degradation processes in tetragonal lysozyme crystals on a silicon substrate by high-resolution X-ray diffractometry

The results of an in situ study of the growth of tetragonal lysozyme crystals by high-resolution X-ray diffractometry are considered. The crystals are grown by the sitting-drop method on crystalline silicon substrates of different types: both on smooth substrates and substrates with artificial surface-relief structures using graphoepitaxy. The crystals are grown in a special hermetically closed crystallization cell, which enables one to obtain images with an optical microscope and perform in situ X-ray diffraction studies in the course of crystal growth. Measurements for lysozyme crystals were carried out in different stages of the crystallization process, including crystal nucleation and growth, developed crystals, the degradation of the crystal structure, and complete destruction.     

Small-angle X-ray scattering study of conditions for the formation of growth units of protein crystals in lysozyme solutions

The structural composition of lysozyme solutions favorable for the formation of the tetragonal form of protein crystals was studied by synchrotron-based small-angle X-ray scattering depending on the protein concentration and the temperature. Along with lysozyme monomers, dimers and octamers are found in crystallization solutions; the octamer content increases with an increase in the protein concentration.     

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

We report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion‐like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino‐acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre‐existing protein crystalline substrates. Tetragonal crystals of hen egg‐white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice‐matched to the ‘host’ apoferritin crystals; this led to mere aggregates of different crystals. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Small-angle X-ray scattering study of the influence of solvent replacement (from H<Subscript>2</Subscript>O to D<Subscript>2</Subscript>O) on the initial crystallization stage of tetragonal lysozyme

The composition of lysozyme solutions in D₂O under conditions favorable for the formation of tetragonal crystals has been investigated at different protein concentrations by small-angle X-ray scattering using the synchrotron radiation. In addition to lysozyme monomers, dimeric and octameric species are found in the crystallization solutions; the octamer content increases with an increase in the protein concentration. A comparison of the data with those obtained under similar conditions but with H₂O used as a solvent has shown that the replacement of light water with heavy one leads to increase of octamer volume fraction in solution.     

Simplex optimization of protein crystallization conditions

Simplex algorithms have been used to optimize for size, number and morphology of lysozyme and apoferritin crystals. This approach requires fewer experiments than the single-factor-at-a-time method or factorial designs and will be useful in conserving materials on the International Space Station. The simplex method has the possible advantage that it conserves on materials by reducing the number of experiments required to optimize a crystallization system. The process is iterative and exploratory and should allow optimum microgravity conditions to be determined which might very well be different from the optimum conditions on Earth. Because the simplex method uses simple mathematical operations to calculate the next set of crystallization conditions it will be easier for crystal growers to implement than factorial designs. Factorial experiments are based on varying all factors simultaneously at a limited number of factor levels. This results in a model that is used to determine the influence of each factor and their interactions. Factorial design experiments are especially useful at the beginning of an experimental study and as a screening tool to investigate a large number of factors. The simplex method is an optimization method which is model-independent and requires no fitting of models to data. Also, when applied to protein crystal growth the simplex method does not rely on an absolute quality score. Instead, with each iteration a comparison is made to the last experiment and the results are assigned as being “better or worse”. In this study, commercially obtained apoferritin was purified from 65% monomeric apoferritin to 92% monomeric apoferritin by size exclusion chromatography. Simplex optimization found the best apoferritin crystals were obtained at 15 mg/ml apoferritin, 2.0% CdSO₄, 25°C using the hanging drop vapor diffusion method of crystallization and at 24 mg/ml apoferritin, 1.5% CdSO₄, 25°C using the containerless crystallization method. For lysozyme, the simplex method found the best crystals at 19 mg/ml lysozyme, 7.0% (w/v) NaCl, pH 4.0, 25°C using the hanging drop vapor diffusion method of crystallization. For both proteins, the optimum conditions were found with less than ten experiments using very little protein. Finally, we report that the factors to be considered in the successful application of this method to crystallization are the number of variables to be studied, the initial conditions, step size and analysis of crystal quality.     

Solubility of tetragonal and orthorhombic lysozyme crystals under high pressure

Two-beam interferometry was applied to measure lysozyme solubility under high pressure. This rapid method allowed determination of one data point within 3 h. The solubility of tetragonal lysozyme crystals was determined as a function of temperature at 0.1, 50, and 100 MPa, and that of orthorhombic crystals was measured at 0.1 and 100 MPa. The solubility of tetragonal crystals increased with pressure; however, that of orthorhombic crystals decreased. In both cases, the enthalpy and entropy of dissolution decreased with pressure.     

A more clear insight of the lysozyme crystal composition

Crystallization can be used as a purification method for proteins. Lysozyme was chosen as a model substance. Changing crystallization conditions will lead as shown to different lysozyme crystal morphologies with different properties. Beside others, lysozyme crystals can show a Tetragonal, High Temperature and Low Temperature Orthorhombic crystal morphology. Experiments such as conductivity measurements, pH tests, chloride detection tests, experiments using methylene blue as a dye and dissolution experiments were carried out to investigate the composition of the lysozyme crystals. It is proven that lysozyme crystals are made up of the initial buffer solution components: lysozyme (the protein), water which is part of the crystal lattice, salt ions which are attached to the protein molecule and voids filled with the buffer solution containing the crystallization agent (e.g. salt). Interesting dissolution behaviours of the lysozyme crystals were observed which are not described so far elsewhere (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

On the incorporation mechanism and distribution mode of impurities in potassium acid phthalate crystals grown from aqueous solutions

Mechanism of impurity structure formation in crystals grown from aqueous solutions has been studied on the example of potassium acid phtalate (abbreviated hereafter as KAP) single crystals. Gold decoration technique at an electronmicroscopic scale has been applied to the study of the distribution of uncontrolled impurities on KAP cleavage face (010) after 10, 20 and 30 days of growth, taking into consideration different growth rates in 〈001〉 and 〈001 〈 directions. A technique for visualization of impurities in water, based on the adsorption of these impurities by the surface of amorphous film of nitrocellulose (parlodion) and the vacuum decoration with gold of these impurities, has been developed. Differences in the impurity structure of KAP regions located in 〈001〉 and 〈001〉 directions from the seed have been established. In 〈001〉 direction after 20 days of growth impurity assemblies 0.1—0.4 μm in size are revealed, and in 〈001〉 direction heterogeneous impurity structure is revealed only after 30 days of growth. The real (impurity) structure of KAP outside impurity assemblies is quite homogeneous and is the same throughout the whole crystal volume, the impurities incorporating mainly into complex active centres. From comparison of the changes in time of the impurity structure of water used for crystallization solutions and the impurity structure of KAP crystals a conclusion is made that the impurity structure of crystals is “programmed” in the impurity structure of crystallization solutions which regularly changes with time, i. e. impurities from different kinds of assemblies which are selectively adsorbed by the growing crystal faces. The role of the adjacent to the growing face interfacial layers which control the growth rate and have a complex impurity structure is stressed.     

Protein crystal growth on the Russian segment of the International Space Station

Experiments on protein crystallization on the Russian segment of the International Space Station were started in 2005. These experiments were performed in the Modul’-1 protein crystallization apparatus specially designed for crystal growth by the free-interface-diffusion method. This paper describes experiments on the crystallization of lysozyme, carboxypeptidase B, and recombinant human insulin on Earth and in microgravity using the Modul’-1 protein crystallization apparatus during the ISS-11-ISS-14 space flights. Crystals of all proteins grown in microgravity have larger sizes than those grown on Earth. Space-grown crystals of lysozyme and insulin characterized by X-ray diffraction were shown to diffract to higher resolution than the Earth-grown crystals. The three-dimensional structures of Zn-insulin crystals grown both on Earth and in microgravity were established. The conformation of the Zn-insulin hexamer in the crystalline state is described.     

Crystalline quality of lysozyme crystals grown in agarose and silica gels studied by X-ray diffraction techniques

The crystalline quality of hen egg white lysozyme (HEWL) crystals grown in agarose gels and in silica gels has been characterized by measuring resolution and mosaic spread. These crystals have been compared to solution grown ones. A quasi-plane-wave X-ray topography study has also been done on some crystals. The study concerns triclinic, monoclinic, orthorhombic and tetragonal forms of HEWL. One observes that the resolution is not really changed by gelling the growth medium, even for rather high gel contents (agarose 0.5% wt/wt). On the contrary, mosaicity, characterized through reflection profile recordings allows to differentiate crystals grown by different techniques: agarose gel grown crystals are, on average, better than solution grown ones but the best crystals are obtained in silica gel. X-ray topography confirms this result.     

Lysozyme crystallization in a precision-controlled temperature gradient

The possibility to control the crystallization process of biomaterials by the temperature was examined by the mathematical simulation of lysozyme crystallization. The precision-temperature control with creation of a local temperature gradient in solution was demonstrated to allow the growth of lysozyme crystals with high structural perfection.     

Solvent freeze out crystallization of lysozyme from a lysozyme‐ovalbumin mixture

Hen egg white lysozyme (HEWL) crystallization conditions from an ovalbumin‐lysozyme mixture were found by screening tests and further located in pseudo‐phase diagrams. This information was used to set up the initial conditions for the solvent freeze out (SFO) process. The process uses the freezing of ice to create the supersaturation for the proteins to crystallize out of the solution. The crystallization of HEWL (15 mg/mL) out of a lysozyme‐ovalbumin mixture (1.7 mg/mL) is carried out by SFO. Under the reported conditions, a crystallization yield of 69 % was obtained. A mean crystal size of 77.8 µm was enhanced in a crystallization time of 15.1 h. The lysozyme nature of the crystals is proven by SDS PAGE and enzymatic activity tests. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

低余辉碘化铯闪烁晶体的生长与性能研究

低余辉碘化铯晶体的开发对于碘化铯晶体在现代安检及医疗CT设备中的应用具有十分重要的意义。本文首先通过原料纯化处理,去掉对晶体余辉有明显影响的杂质,然后采用共掺杂的方式,以改进的布里奇曼法生长低余辉碘化铯晶体,研究了该晶体的闪烁性能。研究结果表明,该方法获得的碘化铯晶体的余辉值约0.31%@50 ms,远低于常规碘化铯晶体的余辉值。     

Influence of impurity on rhythmic crystallization of calcium carbonate in agar gel

During the gel growth of calcite crystals in test tubes, for a narrow concentration of inner and outer electrolytes we observed rhythmic crystallization of calcium carbonate. In this paper the effect of adding impurities on the pattern of periodic crystallization of calcium carbonate has been presented. The addition of impurities either increases or decreases the solubility of reaction product and hence may affect the rate of nucleation of calcium carbonate crystals. The variation of spacing coefficient and velocity constant due to the addition of impurities has also been studied.     

The Properties of Undoped InP and GaP Bulk Crystals Prepared by Floating Zone Melting

Undoped GaP and InP crystals which are produced by floating zone melting without a crucible were investigated. The background concentration was 10¹⁵cm⁻³ after several passages of the molten zone. The fundamental residual impurities are carbon and silicon penetrating into the crystals during the crystallization process. After purification, the electron mobility of GaP was 200 cm²/V. s, and for InP – 4000 cm²/Vs. The compensation decreased to 20%. A very effective purification was observed with respect to the carbon atoms, but for silicon this result is observed only after several passages of the molten zone. GaP and InP are basic materials for optoelectronic devices and it is important to investigate the possibility for production of pure bulk crystals. A proper method for this aim is the floating zone melting, where the crucible effect is removed and the influence of residual impurities is observed. In this work the influence of the number of floating zone passages on the electrical and luminescence properties of InP and GAP bulk crystals has been investigated.     

Implementation of Temperature-Controlled Method of Protein Crystallization in Microgravity

An experimental scientific equipment for implementing temperature-controlled protein crystallization in capillaries under microgravity has been developed, fabricated, and tested. This crystallization method, providing on-line separate control of crystal growth both in the stage of nucleation of crystals and during their further growth, requires small amounts of protein solution. The equipment has been tested on board of Foton-M4 spacecraft (growth of lysozyme protein crystals of high structural quality in microgravity) using a cyclogram developed in ground-based experiments. The results obtained have demonstrated efficiency and importance of the developed equipment and method for growing biomacromolecular crystals of high-structural quality.