The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Nucleation of protein crystals

This paper introduces nucleation theory applied to crystallizing protein solutions. It is shown that the classical approach explains the available nucleation data under most conditions used for growing protein crystals for structural studies and for industrial crystallization. However, it fails to explain most experimental data on the structure of the critical clusters. It is also shown that for open systems working out of equilibrium, such as hanging-drop and counterdiffusion techniques, the geometry of the Ostwald-Myers protein solubility diagram and the number, size, and quality of the forming crystals depend not only on supersaturation but also on the rate of development of supersaturation.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

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.     

Studies of crystal growth mechanisms of proteins by electron microscopy

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.     

A novel dialysis procedure for the crystallization of proteins

Various dialysis methods are commonly employed for the crystallization of proteins. Typical procedures include the use of dialysis bags, dialysis buttons or Zeppezauer microdiffusion cells. The general principle involved is that the protein solution is gradually brought to a point of supersaturation by imposing a gradient of ionic strength or organic solvent concentration across the wall of the dialysis membrane. However, in some cases, the imposition of this gradient across the dialysis membrane can result in the formation of a large number of crystal nucleation sites, thereby giving rise to a reduction in the maximum size of the crystals which can be obtained. A novel 'double-dialysis' procedure which incorporates a second dialysis membrane, thus reducing the rate of equilibration in the crystallization experiment, has been developed in our laboratory. The system has been employed successfully on the delta toxin of Staphylococcus aureus resulting in a useful increase in crystal size. A more quantitative analysis of the technique has been carried out on rat liver malic enzyme. The results of a limited series of crystallization trials with this protein have shown that employment of the 'double-dialysis' technique allows a fine control of the rate of crystal nucleation and therefore provides a mechanism for the controlled growth of large crystals.     

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.     

Effect of polyethylene glycol-400 at low concentrations on long-term growth of muscle phosphoglucomutase crystals from concentrated salt solutions.

Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol-400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long-term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG-induced increase in the "solubility" of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute-induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyethylene glycol and beta-octylglucoside on amorphous, salt-induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility-based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described.     

Practical physics behind growing crystals of biological macromolecules

The aim of this review is to provide biocrystallographers who intend to tackle protein-crystallization with theory and practical examples. Crystallization involves two separate processes, nucleation and growth, which are rarely completely unconnected. Here we give theoretical background and concrete examples illustrating protein crystallization. We describe the nucleation of a new phase, solid or liquid, and the growth and transformation of existing crystals obtained by primary or secondary nucleation or by seeding. Above all, we believe that a thorough knowledge of the phase diagram is vital to the selection of starting position and path for any crystallization experiment.     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.     

Effects of high pressure on the solubility and growth kinetics of monoclinic lysozyme crystals.

Average growth rates of the (0 1 0) and (0 1 0) faces (R<0 1 0>) of monoclinic lysozyme crystals were measured in situ under 0.1 and 100 MPa. From the dependence of the growth rates on the lysozyme concentration, we determined the solubility of the crystal as a function of temperature at 0.1 and 100 MPa. The solubility increased with an increase in pressure. From the comparison between the growth rates under 0.1 and 100 MPa at the same supersaturation level, we found that the growth rates of the monoclinic lysozyme crystals kinetically increase with an increase in pressure. Supersaturation dependencies of the growth rates under 0.1 and 100 MPa were well fitted with a two-dimensional (2D) nucleation growth model of a birth-and-spread type. The fitting results suggest that the increase in the growth rates with pressure can be explained by the decrease in the average ledge surface energy of 2D island, the average distance between the kinks on a step and the activation energies in the incorporation processes of solute molecules.     

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.     

Probabilistic approach to lysozyme crystal nucleation kinetics

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.     

Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile

We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.     

The effect of stirring and seeding on the AcPheLeuNH(2) synthesis and crystallization in a reversed micellar system

The present work describes the enzymatic synthesis and simultaneous crystallization of the dipeptide AcPheLeuNH(2) by alpha-chymotrypsin in a reversed micellar system of tetradecyltrimethylammonium bromide (TTAB)/heptane/octanol/carbonate buffer. The low solubility of the dipeptide in the micellar solution led to the formation and growth of needle-like crystals during the synthesis as soon as supersaturation was achieved. The crystallization process then followed a typical pattern, proceeding in three phases: nucleation, de-supersaturation, and re-equilibrium of saturation. Crystallization was followed by visual observation with an optical microscope, and the increase of crystal number and size was confirmed. Experiments showed that the supersaturation concentration decreases with the addition of AcPheLeuNH(2) seeds before the reaction, and also with a decrease of the stirring speed. It was also observed that the increase of both seed concentration and stirring advances the start of crystallization, so that the dipeptide is more quickly removed from solution. The consequent decrease in its loss through hydrolysis causes an increase in its yield. Both stirring and seeding could constitute important generic strategies for promoting crystallization of more soluble dipeptides during their synthesis in similar reversed micellar systems.     

Kinetic analysis of protein crystal nucleation in gel matrix

The effect of agarose on nucleation of hen egg white lysozyme crystal was examined quantitatively using a temperature-jumping technique. For the first time, to our knowledge, the inhibition of agarose during the nucleation of lysozyme was quantified in two respects: a), the effect of increasing interfacial nucleation barrier, described by the so-called interfacial correlation parameter f(m); and b), the ratio of diffusion to interfacial kinetics obtained from dynamic surface tension measurements. It follows from a dynamic surface tension analysis that the agarose network inhibits the nucleation of lysozyme by means of an enhancement of the repulsion and interfacial structure mismatch between foreign bodies and lysozyme crystals, slowing down the diffusion process of the protein molecules and clusters toward the crystal-fluid interface and inhibiting the rearrangement of protein molecules at the interface. Our results, based on ultraviolet-visible spectroscopy, also show no evidence of the supersaturation enhancement effect in protein agarose gels. The effects of nucleation suppression and transport limitation in gels result in bigger, fewer, and perhaps better quality protein crystals. The understandings obtained in this study will improve our knowledge in controlling the crystallization of proteins and other biomolecules.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

Kinetic factors may reshape the dependence of crystal nucleation rate on temperature in protein bulk solution

Here we provide an analysis of primary results obtained from a study of apoferritin crystal nucleation in compositionally invariant bulk solution at constant supersaturation ratio of the protein. The temperature dependence of the stationary crystal nucleation rate in the protein bulk solution is obtained with the help of experimentally determined probability for detection of at least one crystal per solution volume until a given time. The stationary crystal nucleation rate demonstrates unusual behavior with temperature. We emphasize that this is caused by kinetic factors that are often disregarded in the frame of the classical nucleation theory but can certainly affect the nucleation kinetics.     

Mechanisms of homogeneous nucleation of polymers of sickle cell anemia hemoglobin in deoxy state

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells, occurring when HbS is in deoxy state in the venous circulation. Polymerization is known to start with nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation, yet the mechanisms of nucleation of the primary fibers have never been subjected to dedicated tests. We implement a technique for direct determination of rates and induction times of primary nucleation of HbS fibers, based on detection of emerging HbS polymers using optical differential interference contrast microscopy after laser photolysis of CO-HbS. We show that: (i). nucleation throughout these determinations occurs homogeneously and not on foreign substrates; (ii). individual nucleation events are independent of each other; (iii). the nucleation rates are of the order of 10(6)-10(8)cm(-3)s(-1); (iv). nucleation induction times agree with an a priori prediction based on Zeldovich's theory; (v). in the probed parameter space, the nucleus contains 11 or 12 molecules. The nucleation rate values are comparable to those leading to erythrocyte sickling in vivo and suggest that the mechanisms deduced from in vitro experiments might provide physiologically relevant insights. While the statistics and dynamics of nucleation suggest mechanisms akin to those for small-molecule and protein crystals, the nucleation rate values are nine to ten orders of magnitude higher than those known for protein crystals. These high values cannot be rationalized within the current understanding of the nucleation processes.     

Repair of impurity-poisoned protein crystal surfaces

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.