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

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

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.     

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.     

Sugar bioprotective effects on thermal denaturation of lysozyme: Insights from Raman scattering experiments and molecular dynamics simulation

The bioprotective properties of trehalose, sucrose and maltose on lysozyme protein are studied using Raman spectroscopy and molecular dynamics simulation. The temperature dependencies of the α-helical unfolding processes are determined in presence of different sugars and at various concentrations using the amide I band. The energies of stabilization and acting forces are discussed. The problem of the threshold sugar concentration needed to protect proteins is addressed. The results point out that the three sugars exhibit very similar bioprotective behaviors in the investigated temperature and concentration ranges and suggest that the difference between their respective effects arise mostly from the different denaturated states of lysozyme in the three sugar solutions. Indeed, trehalose gives rise to a significantly more important shift of the denaturation temperature T_m and of the Gibbs net free energy Δ(ΔG_ND) of stabilization of lysozyme with respect to the two other sugars.     

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.     

Phase transitions in lysozyme solutions characterized by differential scanning calorimetry

The detailed understanding of the structure of biological macromolecules reveals their functions and is thus important in the design of new medicines and for engineering molecules with improved properties for industrial applications. However, obtaining high-quality crystals of proteins for determining structures is still quite difficult in general, and successful protein crystallization remains largely empirical and operator-dependent. In this work, a microcalorimetric technique has been utilized to investigate liquid-liquid phase separation through measuring the cloud-point temperature T^cloud for high supersaturated lysozyme solution, and the structure formation of lysozyme solution at low concentration. Pronounced heat-flow curves dependent on solution conditions during cooling process have been obtained and analyzed. The implications of calorimetric results are (i) as to lysozyme solution at low concentration, aggregates form and grow into clusters with the increase of supersaturation in the absence of glycerol, while three-dimensional network instead of aggregates maybe form in the presence of glycerol; (ii) with respect to concentrated lysozyme solution, the cloud-point temperature increases monotonically with the concentration of sodium chloride, and is decresed when glycerol is added as additive.     

Modification of the Langmuir–Schaefer method for fabrication of ordered protein films

A modification of the Langmuir–Schaefer method for the fabrication of high-quality protein films on a solid substrate was proposed and applied to lysozyme. The procedure relies on the use of a pre-prepared protein solution, the parameters of which correspond to crystallization conditions. A lysozyme Langmuir monolayer was shown to be formed with the involvement of complexes, namely, dimers and octamers of protein molecules that are present in such protein solutions. These complexes apparently retain the structure after spreading a protein solution onto an aqueous subphase in a Langmuir trough. The thickness of the film after the transfer of the monolayer, which was formed by the proposed procedure, onto a solid substrate corresponds to the diameter of the octamer and this film is dense, continuous, and uniform, as was demonstrated by several methods: X-ray reflectivity, total external reflection X-ray standing wave, and atomic force microscopy. A layer of chloride ions that formed under the Langmuir monolayer was found at the air–protein film interface. This fact confirms an important role of the precipitating agent (chloride ions) in all steps of the formation of lysozyme films.     

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)     

The dissolution phenomenon of lysozyme crystals

Dissolution studies on lysozyme crystals were carried out since the observed dissolution pattern look different from non‐protein dissolved crystals. The Tetragonal, High Temperature and Low Temperature Orthorhombic morphologies, crystallized using sodium chloride, were chosen and the influence of different pH, salt and protein concentration on their dissolution was investigated. An increase in pH and/or salt concentration can modify the dissolution behaviour. The pattern of the crystals during the dissolution process will, therefore, develop differently. Frequently a skeleton like crystal pattern followed by a falling apart of the crystals is observed. The multi‐component character of the lysozyme crystal (protein, water, buffer, salt) as well as “solvatomorphism” gives first insights in the phenomena happening in the dissolution process. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Correlation of second virial coefficients and solubilities useful in protein crystal growth

The osmotic second virial coefficient, B, is a dilute solution parameter, so one may wonder what relationships exist among B and the many facets of protein crystal growth (PCG). In particular, knowledge of how a protein's solubility (S) depends on solution variables such as temperature, pH and ionic strength is very useful for finding optimum conditions for the PCG experiment. In this work, a correlation between the second virial coefficient and protein solubility is shown for ovalbumin and lysozyme solutions. The data presented suggest significant correlation between B and S as a function of crystallizing salt type and concentration, pH and temperature. The data are presented both in graphical and tabular form so that it maybe useful to researchers desiring to develop and test detailed models for protein interactions.     

Second virial coefficient: variations with lysozyme crystallization conditions

Interparticle lysozyme interactions in solution have been studied by small angle X-ray scattering (SAXS) as a function of salt type (NaCl, NaNO₃, NaSCN and NaOAc), salt concentration, and as a function of temperature between 30°C and 10°C. The choice of conditions was made to cover variations from (undersaturated) solutions to (supersaturated) crystallization conditions. The second virial coefficients (A₂) were determined from the X-ray structure factors extrapolated to the origin, as a function of protein concentration. The A₂ values which correspond to lysozyme crystallization conditions were found to be in a range from about zero to −8.0×10⁻⁴ mol ml g⁻², in agreement with previous determinations by other groups. The variations of the second virial coefficient from positive (repulsive interactions) to negative (attractive interactions) were found to follow the efficiency of salts to induce crystallization. The choice of the second virial coefficient as a tool to predict crystallization conditions is discussed.     

Incorporation of impurity to a tetragonal lysozyme crystal

Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton–Prim–Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.     

Why is the osmotic second virial coefficient related to protein crystallization?

A molecular basis is presented for characterizing the osmotic second virial coefficient, B₂₂, of dilute protein solutions, which provides a measure of the nature of protein–protein interactions and has been shown to be correlated with crystallization behavior. Experimental measurements of the second virial coefficient of lysozyme and bovine α-chymotrypsinogen A were performed by static light scattering, as a function of pH and electrolyte concentration. Although some of the trends can be explained qualitatively by simple colloidal models of protein interactions, a more realistic interpretation based on protein crystallographic structures suggests a different explanation of experimental trends. The interactions accounted for are solute–solute excluded volume (steric), electrostatic and short-range (mainly van der Waals) interactions. The interactions depend strongly on orientation, and this profoundly affects calculated second virial coefficients. We find that molecular configurations in which complementary surfaces are apposed contribute disproportionately to the second virial coefficient, mainly through short-range interactions; electrostatic interactions play a secondary role in many of these configurations. Thus molecular recognition events can play a role in determining the solution thermodynamic properties of proteins, and this provides a plausible basis for explaining the observed relationship with crystallization behavior.     

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.     

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.     

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)     

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)     

Atomic force microscopy study of lysozyme crystallization

Crystallization of lysozyme from solutions has been studied by the atomic force microscopy method. The surface morphology and the growth kinetics of several faces of the orthorhombic and monoclinic modifications of lysozyme crystals are considered. The surface images are obtained at molecular resolution. For the (010) face of orthorhombic lysozyme, the phenomenon of the surface reconstruction is established—doubling of the unit-cell parameter along the a-axis. The main growth parameters of lysozyme are determined—the kink density at steps, probabilities of the attachment and detachment of building blocks, the kink and step velocities, and the dependence of the fluctuation in the step position on time.     

Formation of partially ordered organic planar systems based on the in situ control of their structural organization

The possibilities of significantly improving the quality of planar systems based on photoactive porphyrin–fullerene dyads, layers based on cytochrome c and cardiolipin, and lysozyme crystals and films using a complex of in situ X-ray methods and simulation are described. The potential of X-ray phase-sensitive and surface-sensitive methods developed by M.V. Koval’chuk and researchers from his school in monitoring all stages of synthesis of partially ordered organic structure is demonstrated. This approach shows its efficiency for in situ studies: starting from the formation of complexes in solutions up to the growth of protein films and crystals.     

Microfluidic Cell for Studying the Precrystallization Stage Structure of Protein Solutions by Small-Angle X-Ray Scattering

A microfluidic cell has been developed for studying the structure of protein solutions using small-angle X-ray scattering. The use of prefabricated elements, which provide a small sample volume, allows one to adjust the microfluidic cell parameters to specific problems. Tests of lysozyme solutions were performed on a laboratory diffractometer AMUR-K (Institute of Crystallography, Russian Academy of Sciences), BM29 beamline (ESRF, Grenoble), and “DICSY” beamline of the Kurchatov synchrotron radiation source (National Research Centre “Kurchatov Institute”). Experiments showed that, when a 60-μm-thick quartz glass of special purity grade is applied as an X-ray-transparent window, the intrinsic level of cell background scattering in the range of measured angles is lower than that for quartz capillaries, due to which the measurement accuracy can be increased.