Tetragonal Lysozyme Crystals Research Articles

Endowing Ferroelectric Properties of Tetragonal Lysozyme Crystals through C60 Doping

The inherent nonpolarity of tetragonal lysozyme crystals excludes a ferroelectricity response. Herein, we present a demonstration of achieving measurable ferroelectricity in tetragonal lysozyme crystals through C60 doping. Ferroelectric characterizations revealed that C60-doped tetragonal lysozyme crystals exhibited typical characteristic ferroelectric hysteresis loops. Crystallographic structural analysis suggested that C60 doping may induce a reduction in the overall symmetry of tetragonal Lys@C60, leading to the observed ferroelectricity response. Moreover, the introduction of C60 facilitates efficient electron transport inside the crystal and influences the polarization of Lys@C60, further contributing to the observed ferroelectricity response. This work verifies that C60 doping can serve as a simple strategy to bestow novel ferroelectric properties to non-ferroelectric lysozyme crystals, potentially rendering them suitable for biocompatible and biodegradable application in implantable and wearable bioelectronics.

Precipitant-Free Crystallization of Lysozyme and Glucose Isomerase by Drying

Protein crystallization is usually conducted by using precipitants, although the “salting-out” phenomenon is still unclear and complex. Moreover, the addition of precipitants sometimes results in irreversible disordered precipitation of protein molecules. Although precipitant-free lysozyme crystals obtained by centrifugal concentration showed significant changes in three-dimensional structure compared to the structure of salted-out crystals, it was rather difficult to mount crystals from a viscous dense liquid phase after centrifugal concentration, and the quality of the crystals often deteriorated during the mounting process. Here we present novel precipitant-free crystallization methods, which were effective for lysozyme and glucose isomerase. Tetragonal lysozyme crystals were successfully crystallized in a glass capillary simply by drying highly concentrated lysozyme solution in the presence of 0.01 M hydrochloric acid without using any precipitants. Glucose isomerase dissolved in ultra-pure water was also successfully crystallized in hanging drops by drying highly concentrated solution under low-humidity conditions. Oscillation images of the obtained crystals were safely collected without handling; they clearly indicated the crystals had a tetragonal form for lysozyme and an orthorhombic form for glucose isomerase, and their lattice parameters are similar to those of previously reported crystals obtained by salting-out methods.

Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals.

The hydrogen/deuterium (H/D) exchange of main-chain amide hydrogens in the protein that denatured and refolded in deuterated solvent is considered to contain the traces of hydrogen bond cleavages or the exposure to solvent of the buried part of the protein during the denaturing and refolding (denaturing/refolding) processes. Here, we report the H/D exchange behaviors in hen egg-white lysozymes denatured under acidic conditions, basic conditions, and thermal conditions and then refolded in deuterated solvents, using crystallographic methods. The results indicate that the space containing the Trp28 side chain was hardly exposed to the solvent in acidic conditions, but exposed under basic or heated conditions. Moreover, the β-bridges between Tyr53 and Ile58 in strands β2 and β3, which are in a highly conserved region, show some tolerance to changes in pD. The results indicate that crystallographic method is one of the powerful tools to analyze the denaturing/refolding processes of proteins.

Manipulation of protein crystals using a magnetic field by assembling Fe x O y nanoparticles

Manipulation of protein crystals using an external field is a topic of growing interest in several fields, such as X-ray crystallography and crystal processing. The aim of this study was to develop a method for manipulating crystals using a magnetic field by assembling iron oxide nanoparticles inside a lysozyme crystal. Poly(vinylpyrrolidone)-stabilised iron oxide nanoparticles, prepared through pulsed laser ablation in a solution, were preferentially incorporated in the {101} sectors rather than in the {110} sectors of the tetragonal lysozyme crystal, similar to the gold (Au) and platinum (Pt) nanoparticles studied previously. To keep the crystals intact in solutions, the outer surface of the nanoparticle-assembled crystal was coated with a pure lysozyme crystal and the coated crystals were introduced into a solution containing glycerol. The pure lysozyme crystal at the surface of the nanoparticle-assembled crystal is less likely to dissolve compared with the nanoparticle-assembled crystal itself. Additionally, glycerol has a delaying effect on the dissolution of crystals owing to its high viscosity. The authors successfully demonstrated the handling of protein crystals by commercially available needle magnets in solution. This method requires a simple device with a low cost, without any requirement for control conditions and energy, thus facilitating easy and inexpensive handling of the crystal.

Identification of grown-in dislocations in protein crystals by digital X-ray topography

X-ray topography is a useful and nondestructive method for direct observation of crystal defects in nearly perfect single crystals. The grown-in dislocations from the cross-linked seed crystal in tetragonal hen egg-white lysozyme crystals were successfully characterized by digital X-ray topography. Digital X-ray topographs with various reflections were easily obtained by reconstruction of sequential rocking-curve images. The Burgers vector of the dislocation is different from those reported previously. Interestingly, one of the dislocations had a bent shape. The preferred direction of the dislocation line was analysed by the estimated dislocation energy based on the dislocation theory. The dislocation energy can be estimated by the dislocation theory even in protein crystals composed of macromolecules.

The crystallization enthalpy and entropy of protein solutions: microcalorimetry, van't Hoff determination and linearized Poisson-Boltzmann model of tetragonal lysozyme crystals.

During a first-order phase transition, a thermodynamic system releases or absorbs latent heat. Despite their fundamental importance, the heat or enthalpy change occurring during protein crystallization has been directly measured only in a few cases, and the associated entropy change can only be determined indirectly. This work provides an experimental determination and theoretical analysis of the dependence of the molar crystallization enthalpy of lysozyme solutions, ΔHxtal, on the physicochemical solution parameters. Its value is determined directly by isothermal microcalorimetry and indirectly by a van't Hoff analysis of solubility data, which quantitatively agree. This suggests a two-state crystallization process, in which oligomeric intermediates play a minor role. ΔHxtal is found to be negative on the order of few tens of the thermal energy per molecule. It is independent of protein concentration and stirring speed, but weakly depends on salt (NaCl) concentration and solution pH. Assuming that crystals are electrostatically neutral, these trends are explained by a linearized Poisson-Boltzmann theory. In addition, the molar crystallization entropy, ΔSxtal, is analyzed. The dependence of the van't Hoff entropy on salt concentration and pH is captured by the model, complementing the analysis of crystallization thermodynamics.

Temperature Dependence and Anisotropic Effects in the Thermal Properties of Hen Egg-White Lysozyme Crystals

We measured the thermal conductivity (λ) and thermal diffusivity (α) of tetragonal hen egg-white lysozyme (HEWL) crystals by the transient short-hot wire method. The crystals were grown by two different methods: Magnetically levitated crystals were realized with a superconducting magnet, the c-axis of which was perpendicularly orientated in the direction of the wire, and naturally grown crystals realized by the two-liquid method, grown randomly. We confirmed the temperature dependence in both the λ and α properties by measuring the variations in temperature and by statistical analysis. These properties differed slightly depending on the presence or absence of a magnetic field applied during the crystal growth. We hypothesize that the difference originated from the orientation of the crystals caused by the magnetic field. The statistical analyses demonstrated the possibility that asymmetric thermal conduction in the protein crystals provides anisotropic effects of the thermal properties.

Hydrogen/deuterium exchange behavior in tetragonal hen egg-white lysozyme crystals affected by solution state.

Neutron diffraction studies of hydrogen/deuterium-exchanged hen egg-white lysozyme were performed by a joint X-ray and neutron refinement to elucidate the hydrogen/deuterium exchange behavior. Large crystals for neutron work, consisting of molecules that were exchanged before crystallization, were obtained by repeatedly adding protein solution to the crystal batch using deuterated precipitant reagent. There are differences in hydrogen/deuterium exchange behavior compared with previous crystallographic or NMR studies, which could be due to intermolecular interactions in the crystal or to different lengths of exchange period.

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl2 and CuCl2) as precipitants and the three-dimensional structures were determined at 1.35 Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu+2, Ni+2 and Na+1 cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.Communicated by Ramaswamy H. Sarma

Bayesian machine learning improves single-wavelength anomalous diffraction phasing.

Single-wavelength X-ray anomalous diffraction (SAD) is a frequently employed technique to solve the phase problem in X-ray crystallography. The precision and accuracy of recovered anomalous differences are crucial for determining the correct phases. Continuous rotation (CR) and inverse-beam geometry (IBG) anomalous data collection methods have been performed on tetragonal lysozyme and monoclinic survivin crystals and analysis carried out of how correlated the pairs of Friedel's reflections are after scaling. A multivariate Bayesian model for estimating anomalous differences was tested, which takes into account the correlation between pairs of intensity observations and incorporates the a priori knowledge about the positivity of intensity. The CR and IBG data collection methods resulted in positive correlation between I(+) and I(-) observations, indicating that the anomalous difference dominates between these observations, rather than different levels of radiation damage. An alternative pairing method based on near simultaneously observed Bijvoet's pairs displayed lower correlation and it was unsuccessful for recovering useful anomalous differences when using the multivariate Bayesian model. In contrast, multivariate Bayesian treatment of Friedel's pairs improved the initial phasing of the two tested crystal systems and the two data collection methods.

Characterizing the Cytocompatibility of Various Cross-Linking Chemistries for the Production of Biostable Large-Pore Protein Crystal Materials.

With rapidly growing interest in therapeutic macromolecules, targeted drug delivery, and in vivo biosensing comes the need for new nanostructured biomaterials capable of macromolecule storage and metered release that exhibit robust stability and cytocompatibility. One novel possibility for such a material are engineered large-pore protein crystals (LPCs). Here, various chemically stabilized LPC derived biomaterials were generated using three cross-linking agents: glutaraldehyde, oxaldehyde, and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. LPC biostability and in vitro mammalian cytocompatibility was subsequently evaluated and compared to similarly cross-linked tetragonal hen egg white lysozyme crystals. This study demonstrates the ability of various cross-linking chemistries to physically stabilize the molecular structure of LPC materials-increasing their tolerance to challenging conditions while exhibiting minimal cytotoxicity. This approach produces LPC-derived biomaterials with promising utility for diverse applications in biotechnology and nanomedicine.

Crystallization Technique of High-Quality Protein Crystals Controlling Surface Free Energy

The relationship between protein crystal quality and growth kinetics was assessed by measuring the normal growth rates vs supersaturation of the (110) and (101) faces of dislocation-free tetragonal hen egg white lysozyme crystals at three precipitant concentrations, with NaCl as the precipitant. Assuming a two-dimensional birth and spreading nucleation mechanism, an increase in the surface free energy of the step edges was realized with increasing NaCl concentration, as established by decreases in the full width at half-maximum values of X-ray diffraction rocking curves obtained from crystals. These results demonstrate that controlling the surface free energy of the step edges is an important aspect of obtaining high-quality protein crystals. This work also proposes a mechanism for the observed improvements in the crystal quality, based on the reduced incorporation of impurities into the steps during crystal growth.

Microindentation Hardness of Protein Crystals under Controlled Relative Humidity

Vickers microindentation hardness of protein crystals was investigated on the (110) habit plane of tetragonal hen egg-white lysozyme crystals containing intracrystalline water at controlled relative humidity. The time evolution of the hardness of the crystals exposed to air with different humidities exhibits three stages such as the incubation, transition, and saturation stages. The hardness in the incubation stage keeps a constant value of 16 MPa, which is independent of the humidity. The incubation hardness can correspond to the intrinsic one in the wet condition. The increase of the hardness in the transition and saturation stages is well fitted with the single exponential curve, and is correlated with the reduction of water content in the crystal by the evaporation. The saturated maximum hardness also strongly depends on the water content equilibrated with the humidity. The slip traces corresponding to the (11 ̅0)[110] slip system around the indentation marks are observed in not only incubation but also saturation stages. It is suggested that the plastic deformation in protein crystals by the indentation can be attributed to dislocation multiplication and motion inducing the slip. The indentation hardness in protein crystals is discussed in light of dislocation mechanism with Peierls stress and intracrystalline water.

Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

Dislocation-free tetragonal hen egg white (HEW) lysozyme crystals were grown from a seed crystal in a cell. The rates of tetragonal HEW lysozyme crystal growth normal to the (110) and (101) faces with and without a 1-MHz external electric field were measured. A decrease in the typical growth rates of the crystal measured under an applied field at 1 MHz was observed, although the overall driving force increased. Assuming that the birth and spread mechanism of two-dimensional nucleation occurs, an increase in the effective surface energy of the step ends was realized in the presence of the electric field, which led to an improvement in the crystal quality of the tetragonal HEW lysozyme crystals. This article also discusses the increase in the effective surface energy of the step ends with respect to the change in the entropy of the solid.

Crystallization Technique for Strain-free Protein Crystals Using Cross-linked Seed Crystals

In this study, we show using X-ray diffraction (XRD) rocking-curve measurements that local strain in tetragonal hen egg white (HEW) lysozyme crystals can be removed to a large degree by utilizing seed crystals cross-linked with glutaraldehyde. We also find that misorientation between subgrains and subgrain size decrease when employing cross-linked seed crystals, which suggests that subgrain formation in tetragonal HEW lysozyme crystals can be controlled using this technique. The ideal mosaic structure of proteins that enables the collection of accurate integrated intensities of the diffracted waves is also discussed using tetragonal HEW lysozyme crystals as a model protein.

Technique for High-Quality Protein Crystal Growth by Control of Subgrain Formation under an External Electric Field

X-ray diffraction (XRD) rocking-curves were measured for tetragonal hen egg white (HEW) lysozyme crystals grown with and without application of an external electric field, and the crystal quality was assessed according to the full width at half-maximums (FWHMs) of each rocking-curve profile. The average FWHMs for tetragonal HEW lysozyme crystals grown with an external electric field at 1 MHz were smaller than those for crystals grown without, especially for the 12 12 0 reflection. The crystal homogeneity of the tetragonal HEW lysozyme crystals was also improved under application of an external electric field at 1 MHz, compared to that without. Improvement of the crystal quality of tetragonal HEW lysozyme crystals grown under an applied field is discussed with a focus on subgrain formation. In addition, the origin of subgrain misorientation is also discussed with respect to the incorporation of impurities into protein crystals.

Microcrystal delivery by pulsed liquid droplet for serial femtosecond crystallography.

A liquid-droplet injector has been developed that delivers pristine microcrystals to an X-ray irradiation area for conducting serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL). By finely tuning the pulsed liquid droplets in time and space, a high hit rate of the XFEL pulses to microcrystals in the droplets was achieved for measurements using 5 µm tetragonal lysozyme crystals, which produced 4265 indexable diffraction images in about 30 min. The structure was determined at a resolution of 2.3 Å from <0.3 mg of protein. With further improvements such as reduction of the droplet size, liquid droplets have considerable potential as a crystal carrier for SFX with low sample consumption.

Investigation of the Initial Crystallization Stage in Lysozyme Solutions by Small-Angle X-ray Scattering

From the base structure of tetragonal lysozyme crystals, the octamer cluster was selected as the possible element of crystal growth. The proposed model was verified by small-angle X-ray scattering (SAXS) measurements of lysozyme solutions. The results showed a noticeable presence of lysozyme dimers and octamers under crystallization conditions and the total absence of oligomers under conditions where crystal growth was impossible.

Fast GPU-based absolute intensity determination for energy-dispersive X-ray Laue diffraction

This paper presents a novel method for fast determination of absolute intensities in the sites of Laue spots generated by a tetragonal hen egg-white lysozyme crystal after exposure to white synchrotron radiation during an energy-dispersive X-ray Laue diffraction experiment. The Laue spots are taken by means of an energy-dispersive X-ray 2D pnCCD detector. Current pnCCD detectors have a spatial resolution of 384 × 384 pixels of size 75 × 75 μm2 each and operate at a maximum of 400 Hz. Future devices are going to have higher spatial resolution and frame rates.The proposed method runs on a computer equipped with multiple Graphics Processing Units (GPUs) which provide fast and parallel processing capabilities. Accordingly, our GPU-based algorithm exploits these capabilities to further analyse the Laue spots of the sample. The main contribution of the paper is therefore an alternative algorithm for determining absolute intensities of Laue spots which are themselves computed from a sequence of pnCCD frames. Moreover, a new method for integrating spectral peak intensities and improved background correction, a different way of calculating mean count rate of the background signal and also a new method for n-dimensional Poisson fitting are presented.We present a comparison of the quality of results from the GPU-based algorithm with the quality of results from a prior (base) algorithm running on CPU. This comparison shows that our algorithm is able to produce results with at least the same quality as the base algorithm. Furthermore, the GPU-based algorithm is able to speed up one of the most time-consuming parts of the base algorithm, which is n-dimensional Poisson fitting, by a factor of more than 3. Also, the entire procedure of extracting Laue spots' positions, energies and absolute intensities from a raw dataset of pnCCD frames is accelerated by a factor of more than 3.

A view on the aggregation issue in lysozyme crystallization

The aggregation of lysozyme in crystallizing solutions prior to crystal nucleation and during crystal growth has been the subject of numerous investigations over the past two decades. Nevertheless, it remains a controversial rather than a well-recognized phenomenon. In this study, we investigate the growth of tetragonal lysozyme crystals in quiescent solutions at the early crystal growth stage using seven different protein concentrations ranging from 25 mg ml−1 to 55 mg ml−1, 5% NaCl (w/v), 0.1 M sodium acetate buffer (pH 4.0), and constant temperature (22.0 °C). The growth rates of lysozyme crystals are determined by analyzing the time-lapse images of the evolving visible crystal area, recorded at relatively high magnification (600×). The calculated crystal growth rates are then analyzed according to an equilibrium distribution of lysozyme aggregates, which is based on a model two-body process (monomer ↔ dimer ↔ tetramer ↔ octamer ↔ hexadecamer) developed by M. Li et al. (1995). All types of aggregates are considered separately. We found that both octamers and tetramers are probable candidates for crystal growth units and suggested the most voluminous aggregate fraction as strongly influencing the crystal growth process. We also interpret the lysozyme crystal nucleation in the frame of the classical nucleation theory (CNT) and in accordance with the assumed two-body aggregation reaction set. Our results reveal that the tetramer appears to be the least achievable nucleus size across the whole protein concentration range, where classical nucleation could still be an observable phenomenon.