Protein crystallization in low gravity by step gradient diffusion method.

Two-step crystallization experiments were conducted in low gravity employing a liquid-liquid diffusion method in an effort to eliminate problems associated with protein crystal growth under the supersaturating conditions required for nucleation. Experiments were performed in diffusion cells formed by the sliding of blocks on orbit. Step gradient diffusion experiments consisted of first exposing protein solutions in diffusion half-wells for brief periods to initiating buffer solutions of high precipitant concentrations to induce nucleation followed by expoure of the same protein solutions to solutions of lower precepitant concentration to promote growth of induced nuclei into crystals. To avoid convective disturbances that occur when solutions of discrepant densities are interfaced at normal gravity, crystallization of hen egg-white lysozyme and rabbit skeletal muscle aldolase by step gradient diffusion was investigated in low gravity on four NASA space shuttle flights. In general, the largest ctystals of both proteins formed at the highest initiating precipitant concentration used, which is consistent with nuclei formation upon brief exposure to high precipitant concentration, and that these nuclei are competent for sustained growth at lower precipitant concentration. The two-step approach dissociates nucleation events from crystal growth allowing parameters affecting nucleation kinetics such as time, precipitant concentration and temperature of nucleation to be varied separately from conditions used for post-nucleation growth.     

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.     

Numerical and experimental analysis of periodic patterns and sedimentation of lysozyme

This paper deals with experimental investigation, mathematical modelling and numerical simulation of the crystallization processes induced by counter diffusion method of a precipitant agent in a lysozyme protein solution. Novel mathematical strategies are introduced to simulate the experiments and in particular to take into account the kinetics of the growth process and the motion of the crystals due to the combined effect of gravitational force and viscous drag if the sedimenting process is allowed (protein chamber free of gel). Comparison between experimental observations and numerical simulations in the presence of convection and sedimentation and without them provides a validation of the model. The crystal formation in gel results modulated in space. If the gel matrix is not present, convective cells arise in the protein chamber due to local inversions in the density distribution associated to nucleation phenomena. As time passes, these vortex cells migrate towards the top of the protein chamber exhibiting a different wave number according to the distance from the gel interface. The sedimentating particles produce a wake due to depletion of protein from the surrounding liquid. The models and the experiments may represent a useful methodology for the determination of the parameters and conditions that may lead to protein crystallization.     

Rapid sonocrystallization in the salting-out process

Power ultrasound is applied as a mechanical aid to blend and cavitate the medium during the process of salting-out crystallization. Immediately following the addition of the total precipitant needed to the solution, the ultrasound is turned on. The sonoprocess including nucleation and crystal growth is completed in seconds. The experimental results indicate that the variation of ultrasonic energy, duration or mixture volume can be used to yield advantageous control of the mean size and size distribution of resulting crystals. The crystals insonated have perfect shape and show little agglomeration. Since crystallization in such highly supersaturated solutions is uncontrollable by conventional methods, the insonation plays an irreplaceable role in the process. Based on our experiment, it should be feasible and valuable to scale up this sonoprocess for industrial application.     

In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

The molecular state of hen egg white lysozyme in solution has been studied by small-angle X-ray scattering (SAXS) combined with molecular simulation. The addition of a precipitant is shown to change the state of the protein molecules in solution. The SAXS data were processed using the constructed models of different oligomers. Under the crystallization conditions, lysozyme is shown to be present in solution as monomers (96.0%), dimers (1.9%), and octamers (2.1%), whereas tetramers and hexamers are not found. The modeled structure of the octamer is not consistent with the commonly accepted unit cell containing eight lysozyme molecules. Meanwhile, the modeled octamers are well-fitted to the crystal structure and can serve as building blocks in the course of crystal growth.     

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)     

Spatiotemporal protein crystal growth studies using microfluidic silicon devices

Fundamental investigations of protein crystallization using miniaturized microfluidic silicon devices were presented towards achieving spatiotemporal nucleation and subsequent post-nucleation growth. The developed microfluidic silicon device was typically composed of crystal growth cell, reservoir cell, and optionally of heater elements for supersaturation control. A specific fine pattern area in the growth cell which was fabricated on the silicon substrate with doped p- and n-type silicon layers, served as spatially selective nucleation site of dissolved protein molecules through electrostatic attractive force. In a model material, hen egg white lysozyme, a large number of crystals were grown on the defined nucleation site evenly spaced from each other, whereas parasitic crystal growth positioned around the selective nucleation site, was suppressed by the effects of electrostatic repulsive force between the doped silicon surface and charged protein molecules. A possible crystallization mechanism of describing the heterogeneous nucleation during the initial stage and during the growth of the crystal at the electrolyte–semiconductor silicon surface is proposed and discussed.     

Heterogeneous versus bulk nucleation of lysozyme crystals

Heterogeneous (on‐glass) protein crystal nucleation was separated from the bulk one in systems of thin protein solution layers, confined between two glass plates of custom made quasi two‐dimensional all‐glass cells, as well as by applying forced protein solution flow. Two commercial samples of hen‐egg‐white lysozyme, Seikagaku and Sigma were used as model proteins. Applying the classical technique of separation in time of nucleation and growth stages with protein solution layers of thickness 0.05 cm we found that the on‐glass crystal nucleation prevailed highly with Seikagaku HEWL, while on the opposite, bulk nucleated crystals represented the main crystal fraction in Sigma solution. Also using 0.05 cm solution layers nucleation rates were measured separately for the on‐glass and bulk protein crystals. The process was investigated by varying solution layer thicknesses as well, from 0.05 down to 0.01, 0.0065 and 0.002 cm. Studying the influence of the forced protein solution flow on HEWL crystal nucleation the classical double‐pulse technique was modified by separating the nucleation and growth stages not only in time, but simultaneously also in place. In this case we found that the ratio of on‐glass formed crystal nuclei to bulk nuclei depended on the flow velocity, but in different manner with Seikagaku HEWL and Sigma HEWL. A plausible explanation of our experimental results is that the bulk crystal nucleation occurs on foreign surfaces as well, e.g. on rests of source biomaterial, which are always present in the protein solutions. Moreover, biomaterial seems to be more active nucleant than glass. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Kinetics of protein crystal nucleation and growth in the batch method

We have applied the Johnson–Mehl–Avrami–Kolomogorov (JMAK) theory of crystal nucleation and growth to the problem of protein crystallization in the batch method. Without integrating the JMAK equation explicitly, we use dimensional analysis to derive a general formula for the half-life for decay of the protein supersaturation. This formula includes a dimensioned group and an arbitrary dimensionless function. We integrate the JMAK equation exactly for the special case where the growth rate is independent of the supersaturation and the nucleation rate is proportional to its square. This gives an equation for the time decay of the supersaturation and a formula for the half-life in which all arbitrary dimensionless functions are evaluated. The results are consistent not only with Von Weimarn's rule, which asserts that the average size of a crystal increases as the supersaturation decreases, but also with our experimental results for crystallization of lysozyme, in which the half-life at fixed pH decreases with increasing ionic strength and decreasing temperature.     

Effect of Selected Parameters on Crystallization of Copper Sulphate Pentahydrate

Different batch cooling modes (quick and slow cooling with constant cooling rate, program-med cooling with nearly constant supersaturation) of copper sulphate aqueous solutions have been studied in order to find best conditions for the investigation of the effect of additives on crystallization. Three types of additives (solvents, ionic substances and surfactants) have been used and their effect on crystal size, habit and yield was studied.     

Advancements (and challenges) in the study of protein crystal nucleation and growth; thermodynamic and kinetic explanations and comparison with small-molecule crystallization

This paper reviews advancements and some novel ideas (not yet covered by reviews and monographs) concerning thermodynamics and kinetics of protein crystal nucleation and growth, as well as some outcomes resulting therefrom. By accounting the role of physical and biochemical factors, the paper aims to present a comprehensive (rather than complete) review of recent studies and efforts to elucidate the protein crystallization process. Thermodynamic rules that govern both protein and small-molecule crystallization are considered firstly. The thermodynamically substantiated EBDE method (meaning equilibration between the cohesive energy which maintains the integrity of a crystalline cluster and the destructive energies tending to tear-up it) determines the supersaturation dependent size of stable nuclei (i.e., nuclei that are doomed to grow). The size of the stable nucleus is worth-considering because it is exactly related to the size of the critical crystal nucleus, and permits calculation of the latter. Besides, merely stable nuclei grow to visible crystals, and are detected experimentally. EBDE is applied for considering protein crystal nucleation in pores and hydrophobicity assisted protein crystallization. The logistic functional kinetics of nucleation (expressed as nuclei number density vs. nucleation time) explains quantitatively important aspects of the crystallization process, such as supersaturation dependence of crystal nuclei number density at fixed nucleation time and crystal size distribution (CSD) resulting from batch crystallization. It is shown that the CSD is instigated by the crystal nucleation stage, which produces an ogee-curve shaped CSD vs. crystal birth moments. Experimental results confirm both the logistic functional nucleation kinetics and the calculated CSD. And even though Ostwald ripening modifies the latter (because the smallest crystals dissolve rendering material for the growth of larger crystals), CSD during this terminal crystallization stage retains some traces of the CSD shape inherited from the nucleation stage. Another objective of this paper is to point-out some biochemical aspects of the protein crystallization, such as bond selection mechanism (BSM) of protein crystal nucleation and growth and the effect of electric fields exerted on the process. Finally, an in-silico study on crystal polymorph selection is reviewed.     

Effect of various precipitants on the nucleation rate of tetragonal hen egg-white lysozyme crystals in an AC external electric field

It was observed that the effect of an external electric field on the nucleation rate of tetragonal hen egg-white lysozyme crystals varied depending on the precipitant used (NaCl, NiCl₂ or YbCl₃) and that the electric double layer (EDL) played an important role in generating an external electric field of the necessary strength to control the nucleation rate. This phenomenon depended on the ionic strength of the precipitant used; that is, a precipitant of greater ionic strength resulted in a thinner EDL and increased the effect of the external electric field as the driving force for nucleation. The dependence of the nucleation rate on the precipitant was attributed to the magnitudes of the external electric fields generated in EDLs of varying thickness which were formed in the presence of different precipitants.     

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.     

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.     

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.

     

Kinetics and intimate mechanism of protein crystal nucleation

Experimental and theoretical investigations on protein crystal nucleation are reviewed. Various experimental applications of the classical principle, which requires separation of the nucleation and growth stages of the crystallization process, are described. Temperature control is used most frequently, hypergravity and concentration changes being auxiliary techniques. Nucleation time-lags are measured by imposing temperature evoked supersaturation gradients. Application perspectives are revealed. Nucleation rates are measured according to the classical principle mentioned above, and energy barriers for crystal nucleation and numbers of molecules constituting the critical nuclei are calculated. Surprisingly, although requiring unusually high supersaturation, protein crystal nucleation occurs much more slowly than that with small molecule substances. On this basis novel notions are suggested for the elementary mechanism of protein crystal bond formation. Due to the selection of the crystalline bonding patches a successful collision between protein molecules, resulting in the formation of a crystalline connection, requires not only sufficiently close approach of the species, but also their proper spatial orientation. Imposing a rigid steric constraint, the latter requirement postpones the crystal nucleus formation. Besides, it was shown that cluster coalescence is not a factor, hampering the protein crystal nucleation. The comparison of the model predictions with experimental results proved that nucleation kinetics is governed by kinetic (not by energetic) factors.     

Observation of nucleation effect on crystallization in lithium aluminosilicate glass by viscosity measurement

The crystal nucleation effect in lithium aluminosilicate glasses was investigated by the viscosity measurement with aid of the fiber elongation method. The abrupt increase of viscosity due to the crystallization of glass was observed in viscosity-temperature curve but the minimum viscosity temperature (T_η) related with crystallization showed a strong dependence on the nucleation state such as nucleation temperature, nucleation time and heating rate. The results by viscosity agreed well with those of DTA. The nucleation effect on the microstructure of glass-ceramics was also discussed. Finally, the nucleation effect on the crystallization kinetics was approached quantitatively by calculating the crystal volume from viscosity value.     

AgGa1-xInxSe2晶体的生长习性研究

采用DTA对不同铟含量(x)的AgGa1-xInxSe2多晶熔化和结晶温度进行了测试.结果表明:随着x值的增加其过冷度增大.采用改进的垂直布里奇曼法和实时补温技术,对AgGa1-xInxSe2晶体生长过程中的结晶特性和生长温度场关系进行了研究,并对其结晶形态进行了观测.发现:随着晶体生长过程的进行,熔体结晶温度呈下降趋势,固-液界面发生移动;生长晶体表面存在外形规则、形状相同的半球状小孔,有取向一致的台阶反光面,小孔底部为{112}面.研究结果为大尺寸、高质量的AgGa1-xInxSe2单晶体生长奠定了基础,生长出了尺寸达20 mm×60 mm的完整AgGa1-xInxSe2单晶体.     

添加剂对硝酸钾结晶习性影响的研究

研究了Fe3+、Bi3+、pb2+、EDTA、柠檬酸、季铵盐和十二烷基磺酸钠共7种添加剂对硝酸钾结晶习性的影响,对不同添加剂在晶体表面的吸附机制进行了初步探讨.结果表明,随金属离子Bi3+、pb2+和EDTA浓度的增高,硝酸钾晶体c轴方向生长速度增大,晶体呈棒状;当柠檬酸添加量为100mg/L时,硝酸钾变为长片状;其他添加剂对硝酸钾结晶习性的影响效果则不明显.尽管某些添加剂可以改变硝酸钾的结晶习性,但粉末衍射和单晶衍射分析进一步证明,添加剂并未改变其晶胞参数及结构.     

Understanding protein crystallization on the basis of the phase diagram

The phase diagram of a protein–water system is described with a simple model with parameters for the interaction between the protein molecules in the crystal and in the solution. For a certain range of these parameters the phase diagram shows a metastable liquid–liquid immiscibility region. It is shown that this region corresponds to the “crystallization slot” for growing crystals, as proposed by George and Wilson [Acta Crystallogr. D 50 (1994) 361]. Nucleation in this region proceeds in two steps. First small liquid droplets with a high protein concentration are formed; then small crystalline nuclei grow inside these droplets. In the crystallization slot crystals are covered by a thin liquid film with a high protein concentration. We discuss NMR experiments on lysozyme, which show that nucleation is a transient process with an induction time.