Hen Egg-white Lysozyme Crystals Research Articles

Nucleation-Supersaturation Dual-Drive Crystallization Strategy Enables Efficient Protein Crystallization.

A rational crystallization strategy is essential to obtain high-quality protein crystals, yet the established methods suffer from different limitations arising from the single regulation on either nucleation or supersaturation. Herein, a nucleation-supersaturation dual-driven crystallization (DDC) strategy that realizes synergistic regulation of heterogeneous nucleation sites and solution supersaturation based on dual surface and confinement effects for efficient protein crystallization is reported. This strategy relies on a p(PEGDA-co-DMAA) hydrogel template with pre-filled NaCl under designed concentrations. Once dropping hen egg white lysozyme (HEWL) protein solution on the hydrogel, the wrinkled surface provides numerous nucleation sites, while the internal structure regulates the solution supersaturation in the crystallization region through diffusion. Finally, DDC strategy can create high-quality HEWL crystals with large sizes (100-300µm), well-defined morphologies (hexagon and tetragon), and a significantly accelerated nucleation time (9-12 times faster than that achieved using the conventional hanging drop method). It also performs well at wider protein concentrations (10-50mgmL-1) and categories (e.g., achieving fast crystallization and large-size crystals of trypsin), therefore demonstrating clear advantages and great potential for efficiently fabricating protein crystals desirable for diverse applications.

Spectroscopic Analysis of Concentration Dynamics and Crystallization of Hen Egg-White Lysozyme under Optical Trapping Conditions

Spectroscopic Analysis of Concentration Dynamics and Crystallization of Hen Egg-White Lysozyme under Optical Trapping Conditions

In Situ Observation of Slight Twisting in Hen Egg-White Lysozyme Crystals during Crystal Growth and Dissolution by Digital X-ray Topography

In Situ Observation of Slight Twisting in Hen Egg-White Lysozyme Crystals during Crystal Growth and Dissolution by Digital X-ray Topography

Thermal conductivity and thermal diffusivity of lysozyme crystals, the c-axis of which is magnetically oriented along the direction of the probe wire

ABSTRACT To verify the anisotropic effect on the thermal properties of hen egg white lysozyme crystals, we measured the thermal conductivity and thermal diffusivity of lysozyme crystals, the c-axis of which was magnetically oriented along the wire direction. We used the transient short hot-wire method in a horizontally tilted superconducting magnet bore. The crystals were magnetically levitated at the solution interface by the radial component of the magnetic force. The crystals’ thermal properties grown in the horizontal bore were consistently smaller values than those of the crystals grown in the vertical bore and randomly grown crystals without applying magnetic force.

Textile Functionalization by Porous Protein Crystal Conjugation and Guest Molecule Loading

Protein crystals are versatile nanostructured materials that can be readily engineered for applications in nanomedicine and nanobiotechnology. Despite their versatility, the small size of typical individual protein crystals (less than one cubic mm) presents challenges for macroscale applications. One way to overcome this limitation is by immobilizing protein crystals onto larger substrates. Cotton is composed primarily of cellulose, the most common natural fiber in the world, and is routinely used in numerous material applications including textiles, explosives, paper, and bookbinding. Here, two types of protein crystals were conjugated to the cellulosic substrate of cotton fabric using a 1,1′-carbonyldiimidazole/aldehyde mediated coupling protocol. The efficacy of this attachment was assessed via accelerated laundering and quantified by fluorescence imaging. The ability to load guest molecules of varying sizes into the scaffold structure of the conjugated protein crystals was also assessed. This work demonstrates the potential to create multifunctional textiles by incorporating diverse protein crystal scaffolds that can be infused with a multiplicity of useful guest molecules. Cargo molecule loading and release kinetics will depend on the size of the guest molecules as well as the protein crystal solvent channel geometry. Here, we demonstrate the loading of a small molecule dye into the small pores of hen egg white lysozyme crystals and a model enzyme into the 13-nm pores delimited by “CJ” crystals composed of an isoprenoid-binding protein from Campylerbacter jejuni.

Diffusion Coefficient of Intracrystalline Water in Intrinsic Hen Egg-White Lysozyme Crystals Determined by Confocal Raman Spectroscopy.

Protein crystals composed of protein molecules are expected as a novel porous material. They have high porosity, and the knowledge of the diffusion of intracrystalline water is important. In this study, the diffusion coefficient of intracrystalline water in intrinsic hen egg-white lysozyme (HEWL) crystals was determined by a method that combines confocal Raman spectroscopy and air convection with controlled relative humidity. Similar to common porous materials, the drying process of the protein crystals includes three periods: constant-rate drying, falling-rate drying, and equilibrium state. During the falling-rate drying period, the drying rate depends on the diffusion of intracrystalline water in the protein crystal. The gradient of the water content was measured using confocal Raman spectroscopy. The diffusion coefficient of the intrinsic HEWL crystals was determined as 3.1 × 10-7 cm2/s with a water content of 36.3 vol %. The estimated diffusion coefficients of the intrinsic HEWL crystals without cross-linking were in close agreement with those of the cross-linked protein crystals. This study is timely as the knowledge of the intrinsic diffusion coefficient is useful not only for understanding the mechanism of hydration of proteins but also in practical applications such as porous materials, drug binding, and cryoprotectant soaks.

A miniature airlock system to aid the cryo-cooling of protein crystals grown under anoxic conditions

Crystallographic studies of structures and mechanisms of dioxygen-employing enzymes or oxygen-sensitive proteins usually require these molecules to be prepared and crystallized under anoxic conditions in gloveboxes. Handling, cryo-cooling and extraction of these crystals from gloveboxes for X-ray data collection can be a tricky operation, during which samples might be ruined if they are accidentally exposed to atmospheric oxygen. This work reports the design of a new miniature airlock system and describes the associated method in order to simplify these tasks. The system allows flash-cooling of crystals harvested in a glovebox directly into an external liquid nitrogen container via a miniature airlock while maintaining strict anoxic conditions. The method was assessed using hen egg-white lysozyme crystals soaked in dihydroresorufin (an O2 indicator) and with the reduced tRNA-hydroxylase enzyme MiaE.

Existence of twisting in dislocation-free protein single crystals

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/μm which is more than two orders smaller than 10−3 to 104°/μm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

Multi-objective optimization of protein cooling crystallization with morphological population balance models

Multi-objective optimization of protein cooling crystallization is investigated using the morphological population balance (MPB) models for the simulation of crystal shape and size distributions (CSSD). A combined algorithm framework of multi-objective optimization is proposed based on the coupling of MPB and NSGA-II methods. For the seeded crystallization of hen egg-white (HEW) lysozyme, the Pareto-optimal solutions of six cooling rates are obtained for the combinations of former two and all three objectives about crystal shape, size distribution, and product yield. The relative supersaturation of HEW lysozyme during the crystallization process is found to be much higher than that of common small molecules most likely due to the complex structure of the protein. The crystal shape evolves from the initial rod-like shape into the plate-like shape, resulting from the dominant effect of shape objective. Additionally, incorporating yield objective would considerably enhance the final product ratio.

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.

Growth of gold-aryl nanoparticles in lysozyme crystals

Hen egg-white lysozyme crystals are an exemplary system for the growth of gold-aryl nanoparticles within proteins. We investigated the growth of AuNPs functionalized with carboxyl groups in a single protein crystal of lysozyme (Lys@AuNPs) by the protein reduction of aryldiazonium tetrachloroaurate(III) salt [COOH-4-C6H4NN]AuCl4 over 30, 60, and 90 days. We studied the time-dependent, in situ, and gradual growth of AuNPs within single crystals of lysozyme in precipitant solution of 6.5% NaCl in 0.1 M sodium acetate at pH 4.5. The growth and incorporation of AuNPs inside the lysozyme crystals were confirmed by HR-TEM measurements and microscopy imaging. Investigation of TEM images indicates that AuNPs matured progressively beginning with an average size of 48.5 ± 5.7 nm after 30 days. More numbers of AuNPs of 20.8 ± 4.4 nm and 19.2 ± 5.4 nm after 60 and 90 days, respectively, were formed within the lysozyme crystals. Furthermore, we observed that the addition of Hg2+ can expedite the formation of Lys@AuNPs crystals. The color of the gold nanoparticles was developed after 8 days in the presence of Hg2+. The formed AuNPs were mostly spherical with an average size of 13.9 ± 5.2 nm.

Anthracenyl Functionalization of Half-Sandwich Carbene Complexes: In Vitro Anticancer Activity and Reactions with Biomolecules.

N-Heterocyclic carbene (NHC) ligands are widely investigated in medicinal inorganic chemistry. Here, we report the preparation and characterization of a series of half-sandwich [M(L)(NHC)Cl2] (M = Ru, Os, Rh, Ir; L = cym/Cp*) complexes with a N-flanking anthracenyl moiety attached to imidazole- and benzimidazole-derived NHC ligands. The anticancer activity of the complexes was investigated in cell culture studies where, in comparison to a Rh derivative with an all-carbon-donor-atom-based ligand (5a), they were found to be cytotoxic with IC50 values in the low micromolar range. The Ru derivative 1a was chosen as a representative for stability studies as well as for biomolecule interaction experiments. It underwent partial chlorido/aqua ligand exchange in DMSO-d6/D2O to rapidly form an equilibrium in aqueous media. The reactions of 1a with biomolecules proceeded quickly and resulted in the formation of adducts with amino acids, DNA, and protein. Hen egg white lysozyme crystals were soaked with 1a, and the crystallographic analysis revealed an interaction with an l-aspartic acid residue (Asp119), resulting in the cleavage of the p-cymene ligand but the retention of the NHC moiety. Cell morphology studies for the Rh analog 3a suggested that the cytotoxicity is exerted via mechanisms different from that of cisplatin.

The effects of applying an alternating electric field to lysozyme solutions during the initial crystallization stage

This work investigated the effects of applying low frequency and low intensity alternating current (AC) electric fields on the formation of crystals in protein solutions. The uniform electric fields were applied to hen egg white lysozyme (HEWL) with NaCl in the supersaturated region at room temperature. We evaluated the application time and field intensity dependencies on the number and the size of the generated crystals. Only an hour field application reduced the number of the HEWL crystals. The electric-field applying intensity changed the number of the generated crystals non-monotonically, which shows that the application field intensities have some optimum conditions for the protein crystallization.

Dopamine sheathing facilitates the anisotropic growth of lysozyme crystals

The effect of dopamine (DA) on the nucleation and growth rate of hen egg white lysozyme (HEWL) crystals was studied using polarizing microscopy, powder X-ray diffraction, fluorescence spectroscopy, circular dichroism, and dynamic light scattering, in combination with molecular dynamic simulations. DA increased the solubility of HEWL and promoted the nucleation of HEWL crystals. Crystallization experiments also showed that the shape of HEWL crystals changed from a block to a rod with DA addition, which was attributed to the variation in the growth rates of their neighboring planes. The growth of (101) plane was slightly inhibited and decreased from 3.9 to 3.1 nm/s while the growth rate of (110) decreased significantly from 4.1 to 1.8 nm/s with an increase of DA content. Molecular dynamic simulations indicated that hydrogen bonding and hydrophobic interactions between DA and HEWL were mainly concentrated in the pocket region of HEWL, which enhanced the hydrophilicity and steric hindrance of the area, modulated the assembly behavior of HEWL, and resulted in the anisotropic growth of HEWL crystals. These results suggest that small organic molecules in vivo are an important factor in modulating protein crystal morphology by adjusting the conformational structure of protein molecules.

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.

Analysis of slip systems in protein crystals with a triclinic form using a phenomenological macro-bond method

Slip systems in triclinic hen egg-white lysozyme (Tri-HEWL) crystals, which is one of typical protein crystals, were identified by the indentation method and the surface energy estimation using phenomenological macro-bond methods.

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.

In Situ Absorption and Fluorescence Microspectroscopy Investigation of the Molecular Incorporation Process into Single Nanoporous Protein Crystals.

Protein crystals exhibit distinct three-dimensional structures, which contain well-ordered nanoporous solvent channels, providing a chemically heterogeneous environment. In this paper, the incorporation of various molecules into the solvent channels of native hen egg-white lysozyme crystals was demonstrated using fluorescent dyes, including acridine yellow G, rhodamine 6G, and eosin Y. The process was evaluated on the basis of absorption and fluorescence microspectroscopy at a single-crystal level. The molecular loading process was clearly visualized as a function of time, and it was determined that the protein crystals could act as nanoporous materials. It was found that the incorporation process is strongly dependent on the molecular charge, leading to heterogeneous molecular aggregation, which suggests host–guest interaction of protein crystals from the viewpoint of nanoporous materials.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.