Influence of various factors on the liquid/liquid diffusion crystallization of proteins

From a numerical simulation of pre-nucleation solute transport in the liquid/liquid diffusion crystallization of proteins, it is derived that there are various factors influencing the spatial and temporal distributions of supersaturation. They include the ratio of the length of the protein solution to that of the salt solution in a tube crystallizer, the initial concentrations of protein and salt, and the initial salt concentration in the protein solution. Their influences on the protein crystallization have been demonstrated by the corresponding experiments of gel crystal growth. It may be deduced that the experimental conditions for the liquid/liquid diffusion growth of protein crystals could be optimized under given conditions in order to develop the advantages of this method.     

Modeling of the Biocrystal Growth Using Temperature Field

Crystallography Reports - A mathematical model has been developed and numerical calculations of lysozyme protein crystallization from a homogeneous aqueous solution under temperature field control...     

Supersaturation patterns in counter-diffusion crystallisation methods followed by Mach–Zehnder interferometry

We present experimental observation of the spatio-temporal pattern of supersaturation in counter-diffusion methods. These complex patterns were recorded by dynamical interferometric analysis using a Mach–Zehnder configuration. Tetragonal hen egg white lysozyme crystals were grown inside APCF (advanced protein crystallisation facility) reactors. Salt and protein diffusion profiles were obtained independently by performing duplicated experiments with and without protein in the protein chamber; salt gradients were observed directly while protein concentration profiles are computed from the differences in refractive index between the two experiments. As expected from computer simulations, the time evolution of supersaturation shows a maximum about 45 h after activation (although this value can change as a function of the starting conditions and the geometry of the reactor). Nucleation takes place before this maximum supersaturation is reached. This explains the trend of the growth rate versus time curves for experiments performed within APCF reactors (both on ground and in space) and in capillaries by the gel acupuncture technique. By using very low concentration agarose gel in the protein chamber, sedimentation and buoyancy effects are eliminated so that the effects of gravity on fluid dynamics and hence on the spatio-temporal evolution of supersaturation can be assessed. These results confirm experimentally the predicted behaviour of counter-diffusion systems and support their use in growing large high-quality protein single crystals.     

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.     

Computer modelling of the non-isothermal crystallization kinetics of electroless nickel-phosphorus deposits

Non-isothermal differential scanning calorimetry (DSC) data of electroless nickel-phosphorus (EN) samples with three different phosphorus contents (9, 12 and 16 wt%) has been used in the computer modelling of crystallization kinetics based upon the Johnson-Mehl-Avrami (JMA) theory. The major crystallization processes of the EN samples are complex and could involve multiple reactions, as indicated by the corresponding DSC curves obtained at the heating rates of 5-50 °C/min. The degrees of transformation for major crystallization process of the samples were calculated from those DSC experimental data. Computer simulations of the degree of transformation as well as the corresponding DSC curves were performed using the JMA models, and the results were compared to those from the experiments. It was found that the JMA models could perform within the experimental accuracy of the samples with high (12 and 16 wt%) phosphorus contents. However, satisfactory application of the model to a sample with medium (9 wt%) phosphorus content was not achieved, probably due to the presence of crystalline phase in the deposit prior to crystallization process. The derived JMA kinetic exponents (n) are compared and discussed upon their kinetic interpretation to the non-isothermal crystallization processes of the samples.     

Practical experimental design techniques for automatic and manual protein crystallization

When crystallizers are searching for the optimum crystallization conditions, they often carry out experiments that are confusing and difficult to interpret. This confusion arises because there are several important variables in any protein crystallization experiment (including protein concentration, precipitant concentration, pH and temperature) and these variables often interact–that is to say, changes in the level of one variable often change the optimum settings of the others. Confusion can be avoided by using appropriate experimental designs where all of the important variables are varied in each experimental run. Some well known and practical designs for automatic and manual crystallization are presented, and a simple practical example is given.     

The crystallization of lysozyme and thaumatin with ionic liquid

Three different ionic liquids (ILs) were used for the crystallization of lysozyme and thaumatin. It would be a promising solvent for applications in protein chemistry, due to its unusual features such as excellent solubility of inorganic and organic materials, enhancement of protein activity and stability, etc. Here, [BMIm][BF₄] was used as precipitants or union-precipitants in the experiments of thaumatin and lysozyme, respectively. And the experimental results indicated that the probability of single crystals appearance was significantly increased with the addition of [BMIm][BF₄].     

A short overview on practical techniques for protein crystallization and a new approach using low intensity electromagnetic fields

This contribution deals with a practical overview of some popular and sophisticated crystallization methods that help increase the success rate of a crystallization project and introduces a newly developed method involving low intensity electromagnetic fields. Aiming to suggest a methodology to follow, the present contribution is divided into two main parts in a logical order to get the best crystals for high resolution X-ray crystallographic analysis. The first part starts with a short review of the chemical and physical fundamentals of each crystallization method through different strategies based on physicochemical approaches. Then, practical non-conventional techniques for protein crystallization are presented, not only for growing protein crystals, but also for controlling the size and number of crystals. These include crystal growth in gels, counter-diffusion, seeding, and macromolecular imprinted polymers (MIPs). The second part shows the effects of coupling low intensity electric fields (in the scale of units of  μAmperes) with weak magnetic fields (in the scale of milli Tesla) applied to protein crystallization. This approach consists of a novel experimental set up, which was used to study the influence of the coupled fields on the crystallization of lysozyme in solution and in gel media. This new approach is based on the classical theories of transport phenomena and offers a more accessible strategy to obtain suitable crystals for X-ray characterization or Neutron diffraction investigations.     

Investigation of the possibility of obtaining homogeneous Ga<Subscript>1−<Emphasis Type="Italic">x</Emphasis></Subscript>In<Subscript>x</Subscript>Sb crystals under weak flow conditions

The possibility of growing macrohomogeneous Ga_{1 ? x}In_xSb crystals with x = 0.2 was studied using the axial heating process close to the melt/crystal interface. The grown ingots were analyzed by a JSM-5300 scanning electron microscope and the experimental results were compared with the results of numerical simulation. It was shown that, as a whole, the mathematical model adequately describes the processes of the steady-state heat and mass transfer. It was found that, at the crystallization of Ga_{1 ? x}In_xSb by the axial heating process under conditions of weak laminar flows, longitudinal homogeneity is observed when a dead zone is formed in the melt and that the crystallization regime is similar to diffusion. It was shown that the composition of the grown crystals strongly depends on the structure of a melt flow and its dynamics.     

In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regression model was demonstrated as a monitor of lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrations were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of supersaturation, induction times, and the number and size of the resultant lysozyme crystals. Control experiments were performed by introducing step changes in the reservoir ionic strength. The step changes were initiated by comparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic control of the crystallization process.     

Kinetical study of the nonstoichiometric vapour growth process in the system ZnSe:I2

We have studied the growth of ZnSe crystals by chemical transport in a closed system in nonstoichiometric conditions, and we have deduced that the interface kinetics is the phenomenon limiting the growth process. The effect on the growth process of the deviation from stoichiometry of the II‐VI compound was investigated using a mathematical model that involves indirect data computed from directly obtained experimental values. The experimental crystallization rate was compared with the maximum value of the transport flux calculated using the Arizumi‐Nishinaga model. The influence of the stoichiometry of the source material and of the variations in the growth parameters (supercooling, geometrical dimension, specific loading of the ampoule and iodine concentration) on the ZnSe crystal growth process has also been studied. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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)     

Thermal and chemical methods for producing zinc silicate (willemite): A review

Thermal and chemical methods for producing zinc silicate, Zn₂SiO₄ phosphor on industrial and laboratory scales are reviewed. Zinc silicate has a promising future in advanced materials as a highly versatile luminescent material due to the wide range of multi-colors that can be obtained from various guest ions. Candidates for future industrial methods of producing zinc silicate are critically reviewed from the point of view of phase formation and compared with the conventional solid-state reaction. Conventional methods require calcination at temperatures higher than 1000 °C and long reaction times to form Zn₂SiO₄ phase and these processes limit particle shape and size. Sol–gel methods are performed in a solvent at ambient pressure, while hydrothermal and solvothermal methods tend use high temperatures and high pressures, and especially supercritical water methods tend use conditions higher than 400 °C and 25 MPa. Hydrothermal and sol–gel literature shows that crystallization of Zn₂SiO₄ requires at least temperatures of around 100 °C. Of all the growth methods, supercritical water is able to bring about phase formation in the shortest reaction time. Vapor methods are performed with a gas phase as the reaction medium. Vapor and sol–gel methods require post-calcination for crystallization and have the advantage of providing characteristic particles such as uniform shapes, spherical particles, or nano-sized particles by varying the experimental conditions; they may be combined with the other crystallization routes in the future.     

高岭土水热合成4A沸石实验研究

水热法合成4A沸石的实验是在不同的碱度、不同的液固比、不同的晶化温度和不同的晶化时间条件下进行的,并进行了实验结果和各种合成因素的分析及研究.实验结果分析表明在加碱煅烧及合成沸石的过程中,使用NaOH的效果比使用Na2CO3和KOH,加入适量的晶种能缩短晶化反应时间并能提高合成沸石的质量.在碱度为2.5～4.0M、液固比6∶1～8∶1、晶化温度90～103℃以及晶化时间为3～5h的实验条件下,采用苏州高岭土合成了4A沸石.文中还采用X射线衍射仪和扫描电镜对合成的沸石样品进行了表征.     

Factorially designed crystallization trials of the full-length P0 myelin membrane glycoprotein. I. Precipitation diagram

P0 glycoprotein is the abundant membrane protein of myelin of the peripheral nervous system. We report now the statistical design of the crystallization experiments; based on our belief that important information regarding supersaturation of protein or its solubility nature, as well as metastable state, nucleation or precipitation, are hidden in the trials in which no crystals grow. It is possible to work out this information when the whole set of experiments is designed in such a way as to allow statistical analyses. We selected seven factors, which we believe to be important for crystallization: protein concentration, pH of buffer, nature of precipitant, concentration of precipitant, nature of detergent, additives and temperature. The experimental matrix and detailed work sheet to make 148 solutions having random but balanced combination of these levels were calculated using the program DESIGN. A visual evaluation of crystallization drops was performed using light microscopy. We were able to plot the precipitation boundary diagram. Based on this diagram we have eliminated factors (and levels) that were driving the protein into precipitation. It is known that the precipitation boundary is related to the solubility curves for protein crystals, in the neighborhood of which nucleation and further crystallization is most likely to occur. These conditions are currently being refined to identify important factors (or its levels) that can be crucial in obtaining large and well diffracting crystals. Full-length P0 protein has never been crystallized for structural determination.     

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.

     

Protein crystal nucleation: Recent notions

The nucleation of protein crystals is reconsidered taking into account the specificity of the protein molecules. In contrast to the homogeneous surface properties of small molecules, the protein molecule surface is highly inhomogeneous. Over their surfaces proteins exhibit high anisotropic distribution of patches, which are able to form crystalline bonds, the crystallization patch representing only a small fraction of the total surface of the protein molecule. Therefore, an appropriate spatial orientation of the colliding protein molecules is required in order to create a crystalline cluster. This scenario decreases considerably the success ratio of the attempt frequency for crystal nucleation. On the other hand a heterogeneous nucleation of (protein) crystals may be accelerated due to the arrival on some support of under‐critical clusters that are formed in bulk solution; when arriving there they may acquire the property of critical nuclei. Thus, a plausible explanation of important peculiarities of protein crystal nucleation, as inferred from the experimental data, is suggested. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

DTA peak shift studies of primary crystallization in glasses

Following the recognition during the last decade that knowledge of the shift in the exothermic crystallization DTA peak as a function of pre-DTA isothermal heat-treatment times and temperatures, can provide quantitative information about the crystallization kinetics, there has been renewed interest in DTA investigations of crystallization of glasses. Most studies to date, however, have focussed on the kinetics of polymorphic crystallization (where the compositions of the crystal and the parent glass are the same). These studies have established that the DTA peak shifts to lower temperatures with increased pre-DTA heat-treatment times and JMAK-based formalisms have been developed to extract the steady state nucleation rate from the DTA peak shift data. In this paper, we report new results on the DTA peak shift in systems undergoing primary crystallization (where the compositions of the crystal and glass are different). The DTA results show that the exothermic peak temperature decreases initially but increases later on, becoming significantly larger than the initial value, with increase in the pre-DTA heat-treatment time at a fixed temperature. This increase at long times has not been reported previously and is qualitatively different than the monotonic decrease reported for polymorphic crystallization. To rationalize these new results, a model of primary crystallization has been developed which includes homogeneous nucleation, diffusion-controlled growth, Gibbs-Thomson effect, and a mean field soft-impingement correction during growth. Based on this model and experimental results, it is concluded that the initial shift to lower temperatures is due to an increase in the number of nuclei (as concluded previously by others for the case of polymorphic crystallization) and the later shift towards high temperatures in our experiments is due to diffusion-controlled growth during the pre-DTA heat treatment.