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)     

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)     

Growth of rhombohedral insulin crystals and in vitro modeling of their dissolution in the blood stream

Insulin is the only protein that is secreted in a crystalline form in a human healthy body. To mimic the secretion process we used NaCl salting‐out to growing tiny rhombohedral Zn‐insulin crystals. The dissolution of the insulin crystals is of special interest for the therapeutical praxis, because the human body is supplied with the physiologically active monomers of the insulin through dissolution of the crystalline granules secreted in the pancreatic β‐cells. Sets of tiny rhombohedral Zn‐insulin crystals, which resembled the granules secreted in the β‐cells, were subjected to dissolution in blood plasma and model solutions. The impacts of the solution composition, flow rate, pH and ionic strength on the insulin crystal dissolution were investigated. The effect of the blood plasma was determinant because it dissolved the rhombohedral Zn‐insulin crystals almost instantly, while the effects of solution's physicochemical characteristics were of minor importance. In addition, we found that the presence of abundant zinc ions suppressed the dissolution of the insulin crystals. (© 2011 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)     

Brittleness of protein crystals

A simple technique for studying the brittleness of small crystals is reported. The limits of fracture toughness of tetragonal hen‐egg white lysozyme crystals, oriented with their c‐axis normally to the substrate, were measured. The strong mechanics anisotropy of those crystals was confirmed. The role of the water present in the protein crystal lattice was re‐considered in seek for a more holistic understanding of the process, the idea being that the intra‐crystalline solution sustains the globular protein molecules in their native configuration. Also it is argued that this water may contribute for holding together the huge bio‐molecules in the crystal lattice (that is to act as additional “glue” in the crystal). The hypothesis is that dynamic chains of H‐bonds in the intra‐crystalline water are likely to be prolonged to connect protein‐to‐water‐to‐protein.     

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)     

Batch crystallization of soluble proteins: effect of precipitant, temperature and additive

Although techniques used for protein crystallization have been progressing greatly, successful crystallization is still largely empirical and operatordependent. The crystallization of biological macromolecules is a pretty complicated process involving numerous parameters, thus the detailed understanding of the effect of crystallization conditions on macromolecule crystallization is advantageous. In this study, we have investigated the effect of precipitant, temperature, and additive on the crystallization of lysozyme and chymotrypsinogen A. As the precipitant, sodium chloride is more effective to the crystallization of lysozyme, and ammonium sulfate is more suitable to the crystallization of chymotrypsinogen A. Temperature is found to have no effect on the crystal habit of chymotrypsinogen A, while lysozyme crystallization displays highly sensitive temperature dependence, gradually varied temperature can result in better crystal habit and quality of lysozyme crystals. Furthermore, non-electrolytic additives dimethyl sulfoxide (DMSO) and glycerol are found to not only to increase the protein's solubility, but also decrease the critical supersaturation S_c for explosive nucleation of highly supersaturated protein solution. It is suggested that these additives can affect the interactions between protein molecules, thermodynamic equilibrium, surface energy of the crystal, and nucleation process of protein crystallization.     

The distribution of impurities in lysozyme crystals

The effective distribution coefficient, K_Ieff, of turkey egg white lysozyme into host hen egg white lysozyme crystals was measured for varying levels of relative supersaturation, impurity concentrations and temperatures. Turkey egg white lysozyme served as a test impurity of high homology to hen egg white lysozyme. K_Ieff increased with increasing host protein concentration and temperature, consistent with a kinetically controlled incorporation process. The principles of slow crystal growth rate to limit impurity incorporation were applied to a commercial source of lysozyme with concomitant improvement in purity of re-crystallized protein.     

Sedimentation as a tool for crystallization from protein mixtures

Crystals from apoferritin which is an iron‐free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO₄ in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (∼1,14µM/L) in 25mg/ml (∼1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (∼1:10³) of two proteins is used to stress on the observed effect. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

X-ray diffraction studies of protein crystal disorder

Protein crystals contain many kinds of disorder, but only a small fraction of these are likely to be important in limiting the diffraction properties of interest to crystallographers. X-ray topography, high-angular-resolution reciprocal space measurements, and standard crystallographic data collection have been used to probe three factors that may produce diffraction-limiting disorder: (1) solution variations during crystal growth, (2) macromolecular impurities, and (3) post-growth crystal treatments. Variations in solution conditions that occur in widely used growth methods may lead to variations in equilibrium protein conformation and crystal packing as a crystal grows, and these may introduce appreciable disorder for sensitive proteins. Tetragonal lysozyme crystals subjected to abrupt changes in temperature, pH, or salt concentration during growth show increased disorder, consistent with this mechanism. Macromolecular impurities can have profound effects on protein crystal quality. A combination of diffraction measurements provides insight into the mechanisms by which particular impurities create disorder, and this insight leads to a simple approach for reducing this disorder. Substantial degradation of diffraction properties due to conformation and lattice constant changes can occur during post-growth crystal treatments such as heavy-atom compound and drug binding. Measurements of the time evolution of crystal disorder during controlled crystal dehydration – a simple model for such treatments – suggest that structural metastability conferred by the constraints of the crystal lattice plays an important role in determining the extent to which the diffraction properties degrade.     

Growth and dissolution of equally‐sized insulin crystals

A protocol for growing sets of nearly uniform size crystals was devised and tested experimentally. The experiments were centered on insulin because of its medical significance however the method is applicable to other substances as well (C.N. Nanev, V.D. Tonchev, F.V. Hodzhaoglu, Protocol for growing insulin crystals of uniform size, J. Cryst. Growth 375 (2013)10–15). Now, both growth and dissolution of equally‐sized crystals are described quantitatively by a common analytical model. In our model the emphasis is put on the dissolution case when crystals number and/or size are sufficiently large to secure reaching solubility, while some non‐dissolved crystalline substance is still remaining. Quantitative results are obtained for the relations between dimensionless values of crystal size, solution concentration and time elapsed, the assumption simplifying our calculations being that the crystals retain their shape during the entire dissolution process.     

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.     

Study of the crystal shape and its influence on the anti‐tumor activity of tumor necrosis factor‐related apoptosis‐inducing ligand (Apo2L/TRAIL)

This paper describes a method about the crystal shape control improving the anti‐tumor activity of tumor necrosis factor‐related apoptosis‐inducing ligand (Apo2L/TRAIL) in a batch cooling suspension crystallization by selecting pH values as a controllable variable. Three shaped TRAIL crystals could be obtained under different pH conditions, among which the hexagonal plate crystals had the highest specific activities against tumor cell line. The relationship among pH values, trimer contents and the specific activities of crystals and the purified TRAIL solutions were investigated. The results showed that different trimer contents resulted from pH altering in crystals and protein solutions is a main reason for their different specific activities. The studies may supply a new method to improve the bioactivity of TRAIL agent during its production and storage. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Adhesion of protein crystals: Measurement of the detachment force

The degree of adhesion of protein crystals, heterogeneously nucleated and grown on different supports (e.g. glass plates and plates coated with poly‐L‐lysine, hexamethyl‐disilazane and silicon) is measured directly with a purposely‐developed technique. The sticking force crystal/support is determined by means of a flexible glass fibre, which bending is calibrated by means of series of weights. In this way an elastic constant, specific for each glass fiber is determined individually. Appropriate glass fibres with relative bending less than 10% (Hook's law) are used. The force which is necessary to be exerted, by means of a micro‐manipulator, in order to detach the crystal from the support is taken as a quantitative measure for the adhesion strength. Forces between 10 N cm^‐2 and 1 N cm^‐2 for differently oriented tetragonal hen‐egg‐white lysozyme and cubic ferritin crystals, and 0.1 N cm^‐2 for rhombohedral (porcine) insulin and orthorhombic trypsin crystals are measured. The tetragonal HEWL and rhombohedral insulin crystals show anisotropy of the adhesion strength. In contrast, the cubic ferritin crystals are isotropic also in this respect. For comparison purposes adhesion measurements are performed with NaCl and sugar crystals. An attempt is made to evaluate also the adhesion energy of the protein crystals. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spontaneous crystallization of potassium chloride from aqueous and aqueous‐ethanol solutions; Part 3: Model of the crystallization process

A model of spontaneous crystallization process was proposed. The model describes kinetics of the crystallization process after the end of the induction period. To test the model the published earlier data on crystallization and aggregation kinetics of potassium chloride at its spontaneous crystallization from supersaturated aqueous and aqueous‐ethanol solutions were used. It was found excellent coincidence of the experimental and theoretical data on concentration of the salt and the total number of crystals in solution at crystallization. Somewhat change for the worse was at the theoretical calculations of crystal size distribution at the end of the crystallization process. It indicated that the ways of calculation of size of crystals and their weight fraction in deposit were very approximate. The model allows predicting with satisfactory accuracy kinetics of crystallization using such general parameters of potassium chloride as the specific surface energy and the height of the nucleus‐bridges between crystals at coalescence. It needs further test of the model for other salts. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

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.     

Crystallization of chicken egg white lysozyme from assorted sulfate salts

Chicken egg white lysozyme has been found to crystallize from ammonium, sodium, potassium, rubidium, magnesium, and manganese sulfates at acidic and basic pH, with protein concentrations from 60 to 190 mg/ml. Crystals have also been grown at 4°C in the absence of any other added salts using isoionic lysozyme which was titrated to pH 4.6 with dilute sulfuric acid. Four different crystal forms have been obtained, depending upon the temperature, protein concentration, and precipitating salt employed. Crystals grown at 15°C were generally tetragonal, with space group P4₃2₁2. Crystallization at 20°C typically resulted in the formation of orthorhombic crystals, space group P2₁2₁2₁. The tetragonal ↔ orthorhombic transition appeared to be a function of both the temperature and protein concentration, occurring between 15 and 20°C and between 100 and 125 mg/ml protein concentration. Crystallization from 1.2 M magnesium sulfate at pH 7.8 gave a trigonal crystal, space group P3₁2₁, a=b=87.4, c=73.7, γ=120°, which diffracted to 2.8 Å. Crystallization from ammonium sulfate at pH 4.6, generally at lower temperatures, was also found to result in a monoclinic form, space group C2, a=65.6, b=95.0, c=41.2, β=119.2°. A crystal of ∼0.2×0.2×0.5 mm grown from bulk solution diffracted to ∼3.5 Å.