Hen Egg-white Lysozyme Crystals Research Articles

Quasi-Laue neutron-diffraction study of the water arrangement in crystals of triclinic hen egg-white lysozyme.

Triclinic crystals of lysozyme, hydrogen-deuterium exchanged in deuterated solvent, have been studied using neutron quasi-Laue techniques and a newly developed cylinder image-plate detector. The wavelength range employed was from 2.7 to 3.5 A, which gave 9426 significant reflections [F >/= 2sigma(F)] to a resolution limit of 1. 7 A. The deuteration states of the H atoms in the protein molecule were identified, followed by an extensive analysis of the water structure surrounding the protein. The final R factor was 20.4% (Rfree = 22.1%). In total, the 244 observed water molecules form approximately one layer of water around the protein with far fewer water molecules located further away. Water molecules covering the apolar patches make tangential layers at 4-5 A from the surface or form C-H...O contacts, and several water-molecule sites can be identified in the apolar cavities. Many of the water molecules are apparently orientationally disordered, and only 115 out of the 244 water molecules sit in mean single orientations. Comparison of these results with quasi-elastic neutron scattering observations of the water dynamics leads to a picture of the water molecules forming an extended constantly fluctuating network covering the protein surface.

Crystallization of proteins under an external electric field

An external electric field affects the crystallization of proteins when applied under some conditions of temperature, pH, and precipitating agent composition. As suggested in the theoretical part of this paper, it produces large protein concentration gradients inside the mother liquor leading to a local supersaturation area in the crystallization solution. Such an experiment has been used for the first time on the crystallization of a protein. The effects of an external electric field on the crystallization of hen egg-white lysozyme at 293 K, pH 4.5, and two NaCl concentrations (0.6–0.7 M) have been investigated using the vapor diffusion method. The application of electric field results in a smaller number of crystals with larger size. The crystals grew at the droplet surface, near the cathode. The nucleation rate is drastically reduced and this experimental method could be used to control the number of crystals in the droplet.

Two-photon fluorescence imaging of impurity distributions in protein crystals

Macromolecular impurities present in solution have profound effects on the growth and quality of protein crystals used for x-ray structure determinations. We have imaged the three-dimensional distribution of ovotransferrin impurities in crystals of the protein hen egg white lysozyme (HEWL) using two-photon excitation fluorescence microscopy. Impurity concentrations differ between the two types of growth sectors present in tetragonal HEWL crystals and impurities preferentially incorporate along the boundaries between growth sectors. Cracked crystals show large impurity-rich cores that are not observed in uncracked crystals. These nonuniform impurity distributions provide insight into how and why impurities affect crystal quality. Our results have implications for crystal growth and for protein purification.

Growth and mechanical properties of lysozyme crystals

Vickers microhardness of protein crystals of hen egg-white lysozyme was investigated by the indentation method. The indentations were clearly made on the crystal surface and no large elastic recovery of them was observed. The value of the hardness was estimated to be 0.2 kgf/mm 2 on the crystals just after they were transferred from solution to air. The hardness drastically increased with increasing exposure time to air at room temperature. The maximum hardness was 20 kgf/mm 2, which is almost the same as those of van der Waals crystals. The origin of such a change in hardness is explained in terms of the water contained in protein crystals.

Lower dimer impurity incorporation may result in higher perfection of HEWL crystals grown in microgravity: A case study

Crystals of tetragonal hen egg white lysozyme (HEWL) grown on a series of space missions and their terrestrial counterparts were analyzed by gel electrophoresis and X-ray diffraction. The crystals were produced by vapor-diffusion and dialysis methods. The microgravity and terrestrial grown HEWL crystals were found to have effective partitioning coefficients ( K eff) for an oxidatively formed covalent dimer impurity (MW 28 K) of 2 and 9, respectively, i.e. the latter contain 4.5 times more dimers. The microgravity grown crystals allowed the collection of 24% more useful reflections and improved the resolution from 1.6 to 1.35 Å. Other improvements were also noted including lower isotropic B-factors of 16.9, versus 23.8 Å 2 for their terrestrial counterparts. High-resolution laser interferometry was applied quantitatively to evaluate the influence of dimer impurity on growth kinetics. It is shown that the growth of the (1 0 1) face from solution into which 1% dimers were introduced decelerates with increasing solution flow rate and the growth stops at a flow rate of about 0.2 mm/s. This effect occurs faster than in ultrapure solutions. The covalently bound dimers essentially increase the amplitudes of the striation-inducing growth rate fluctuations. The effect is ascribed to the enhanced transport of growth inhibiting HEWL dimer to the interface. Theoretical analysis shows that a stagnant solution around a growing crystal is strongly depleted with respect to impurity by about 60% for the measured growth parameters as compared to the solution bulk. Thus, a crystal in microgravity grows from essentially purer solution than the ones in the presence of convection flows. Therefore, it traps less stress inducing impurity and should be more perfect. For crystal/impurity systems where K eff is small enough microgravity should have an opposite effect.

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.

Crystalline quality of lysozyme crystals grown in agarose and silica gels studied by X-ray diffraction techniques

The crystalline quality of hen egg white lysozyme (HEWL) crystals grown in agarose gels and in silica gels has been characterized by measuring resolution and mosaic spread. These crystals have been compared to solution grown ones. A quasi-plane-wave X-ray topography study has also been done on some crystals. The study concerns triclinic, monoclinic, orthorhombic and tetragonal forms of HEWL. One observes that the resolution is not really changed by gelling the growth medium, even for rather high gel contents (agarose 0.5% wt/wt). On the contrary, mosaicity, characterized through reflection profile recordings allows to differentiate crystals grown by different techniques: agarose gel grown crystals are, on average, better than solution grown ones but the best crystals are obtained in silica gel. X-ray topography confirms this result.

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.

Effect of a magnetic field on the orientation of hen egg-white lysozyme crystals

Recent experiments have shown that lysozyme crystallization in a magnetic field of the order of 1 T can result in a significant degree of orientation of the crystals. We present more extensive experimental results and propose a model to account for this phenomenon. Because of the small susceptibility anisotropy of most protein molecules, the orienting effect is unimportant for smaller aggregates, even those much larger than a critical nucleus. However, during sedimentation crystals grow larger and are more likely to become aligned. The degree of orientation thus depends on crystal growth rate and container geometry, in addition to magnetic field strength, as we have confirmed experimentally.

Heterogeneous nucleation (and adhesion) of lysozyme crystals

The heterogeneous nucleation of hen-egg-white lysozyme (HEWL) crystals has been investigated using a double-(thermal)-pulse technique, thus detaching nucleation from the growth stage. n( t) dependencies of the nucleus number, n, vs. time, t, were plotted for surfaces rendered hydrophobic (by means of hexamethyl-disilazane) and for bare glass surfaces. Preferred crystallite orientation supplied additional information. The discussion is based on the classical Stranski–Kaischew theory of crystal nucleation. With a purposely developed new technique the adhesion energies have been measured for HEWL crystals, grown on different supports.

Estimating lysozyme crystallization growth rates and solubility from isothermal microcalorimetry.

A microcalorimetric technique has been developed to measure crystal-growth kinetics and enthalpies of crystallization. The enthalpy of crystallization of hen egg-white lysozyme in 0.1 M acetate buffer at pH 4.6 was determined at 287 K using this technique. The enthalpies were directly measured to be -14.3 +/- 2.0 and -14.6 +/- 1.3 kcal mol-1 (1 kcal mol-1 = 4.184 kJ mol-1) for 3 and 5% NaCl solutions, respectively, which is in good agreement with values estimated from previous solubility measurements. Non-linear regression of the transient heat flow allowed measurement of the crystal growth rate as a function of protein supersaturation as well as the solubility. The crystal growth rates determined by this method were found to agree with those in the literature under the same solution conditions.

In situ measurement and dynamic control of the evaporation rate in vapor diffusion crystallization of proteins

A special device with a weight-sensitive facility was designed for monitoring and controlling the water evaporation in vapor diffusion protein crystallization. The device made it possible to measure the weight of the drop in real time while the crystallization experiment was going on normally. The precise water equilibration curves under different crystallization conditions could be obtained automatically. By monitoring and controlling the evaporation rate, the crystallization of hen egg-white lysozyme and trichosanthin, a plant protein from Chinese herb, was optimized by regulating the reservoir solution dynamically. The experimental results of these two proteins indicate both the feasibility of the device and the usefulness of dynamic control technique. Compared with traditional crystallization experiments, dynamically controlled crystallization can reduce the number of nuclei, increase the crystal size and save experimental time effectively.

Gel growth of lysozyme crystals studied by small angle neutron scattering: case of silica gel, a nucleation inhibitor

Silica gels were used as a media for crystal growth of hen egg white lysozyme crystals. Using small angle neutron scattering, gel-free solutions have been compared to gelled samples in the pre-nucleation period and in the growth period. Characterisation of the protein solution trapped in the gel network shows an adsorption of protein on the gel surface through electrostatic and most probably through hydrogen bonding interactions. The consecutive decrease of concentration is consistent with the decrease of the nucleation rate with respect to gel-free solutions. By modification of the gel surface, we succeeded in increasing the number of nuclei. Growth occurs by incorporation of free molecules and in a second stage, by incorporation of molecules which are progressively desorbed. This provides a relatively constant concentration of the free molecules.

Gel growth of lysozyme crystals studied by small angle neutron scattering: case of agarose gel, a nucleation promotor

In order to understand why hen egg white lysozyme crystals nucleate more easily in agarose gel than in gel-free solutions, we have made a small angle neutron scattering study of gelled and gel-free lysozyme solutions for different supersaturation conditions by varying the temperature, protein and salt (NaCl) concentrations. For higher supersaturations, some kinetics experiments have been done. We have analysed the SANS signal as resulting from two contributions. A first contribution is due to free protein monomers. It shows that concentration and interactions are identical in gel and gel-free media. In this respect, the gel is neutral and supersaturation is not changed. A second contribution is attributed to lysozyme clusters of several tens to several hundreds of nanometers. This contribution increases by increasing supersaturation and, for a given supersaturation, is noticeably stronger in gel than in solution. This effect being not observed when the gel is not set (agarose sol), it has been related to the mechanical properties of the medium. These considerations lead us to propose a link between clustering and nucleation, namely, nucleation would be built out of some of these clusters.

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

Microgravity in space offers an ideal environment for growing protein crystals, where there is an absence of density-driven convection and sedimentation. Using domestic apparatus of protein crystalizetion, two space experiments of protein crystal growth have been carried out on the Chinese re-entry satellite, and good-quality hen egg-white lysozyme crystals were obtained. To study the effect of microgravity on the prorein crystal structure, three structures of lysozyme crystals grown in gravity and microgravity were determined at high resolution.

Effects of microheterogeneity in hen egg-white lysozyme crystallization.

In earlier sodium dodecylsulfate-polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS-PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with Mr = 28000 and 27500. In addition, larger HEWL oligomers with Mr = 1.7 million and 320000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram-1 mg ml-1 OHEWL to 32 mg ml-1 HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-0.3 M hydrogen peroxide. Repartitioning of the dimer during crystallization at various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained </= 25%(w/w) of the oligomers in the solution, with no apparent temperature dependence of the repartitioning.

A neutron diffractometer for biological macromolecules using neutron imaging plates

Neutron imaging plates (NIPs), developed recently, have a good spatial resolution and availability of a large sensitive area. The NIPs having such characteristics may be particularly useful for neutron-diffraction measurements of biological macromolecules. We have constructed a neutron diffractometer dedicated to crystallography of biological macromolecules using the NIP, in the guide hall of the reactor JRR-3M at Japan Atomic Energy Research Institute. Neutrons are monochromatized with an elastically-bent-silicon monochromator ( λ = 0.22 nm). The diffraction patterns are detected with the NIP of 400 × 520 mm 2 at a distance of 150–300 mm from a sample crystal. A sequence of the measurements (exposure, reading the NIP, erasure of the pattern, and starting the next exposure) is done automatically. We measured the diffraction patterns from hen egg-white lysozyme crystals, and obtained the patterns which can be processed to derive integrated intensity of each diffraction spot.

Magnetic orientation as a tool to study the initial stage of crystallization of lysozyme

The tetragonal crystals of hen egg-white lysozyme align their c-axis in the direction of a magnetic field. By applying the magnetic field of 1.6 T only over some period during the whole crystallization process, it was possible to know when the crystals sedimented. It was found that crystals grew in solution, and started to sediment on reaching a critical size. We evaluated the critical size to be 1–2 μm by changing the magnetic field strength (0.1–1.2 T) and analyzing the relation between the field strength and the proportion of magnetic orientation.

Effects of a magnetic field on the nucleation and growth of protein crystals

Crystallization of hen egg-white lysozyme and horse-spleen ferritin was carried out under a steady and uniform magnetic field of 10 T and compared with the crystals grown under 0 T. It is clearly demonstrated that a magnetic field reduced the number of nuclei and not only oriented the crystals but also modified the habit of protein crystals. The present experimental result indicates that application of a magnetic field is an efficient method for growing a small number of large crystals.

Effect of hydrostatic pressure on the crystallization of lysozyme based on in situ observations

The growth rate ( R) and solubility ( c s) of the tetragonal crystals of hen egg-white lysozyme were determined under hydrostatic pressure up to 350 MPa based on the observations through transparent windows. When the pH is 4.6 and the NaCl concentration 3.0% (w / w), c s (in % (w / w)) increases with pressure p (in MPa) at 23°C approximately as c s = 0.78 + 0.0152 p. In contrast to the linearly increasing c s, R decreased non-linearly with pressure, and was practically zero in a wide pressure range from about 100 to 200 MPa. This means that conventional methods of determining the solubility based on the concentration change are difficult to be used under high pressure. This problem can be overcome by combining in situ observations with image analyses.