Lysozyme Crystals Research Articles

Hydrolase and Peroxidase Activity of Gold Nanocluster Composites with Lysozyme and Cross-Linked Lysozyme Crystals

Hydrolase and Peroxidase Activity of Gold Nanocluster Composites with Lysozyme and Cross-Linked Lysozyme Crystals

The interactions of protein-calcium oxalate in crystallization process

Understanding the pathogenic crystallization of calcium oxalate (CaOx), a major constituent of kidney stones, is crucial in developing effective treatment strategies. In this work, the interactions between lysozyme and CaOx during crystallization have been investigated. Further investigations into the crystal morphology, size, quantity, and nucleation time of the lysozyme have been designed. The results reveal that CaCl2 could act as an effective precipitant for lysozyme crystallization. The formation process and structure of the distinctive composite CaOx-lysozyme crystal has been investigated. The addition of lysozyme promoted the formation of individual CaOx crystals by preventing the crystal aggregation in the solution. The presence of lysozyme induced a transformation in the CaOx crystals from calcium oxalate monohydrate to calcium oxalate dihydrate. These findings contribute to a better understanding of protein-CaOx interactions and their implications in kidney stone pathogenesis.

Crystallization of Lysozyme Induced by Gap-Mode Surface Plasmon Resonance of Gold Nanocolloids

Crystallization of Lysozyme Induced by Gap-Mode Surface Plasmon Resonance of Gold Nanocolloids

Hen egg lysozyme polymorphs mediated by two ionic liquids during batch cooling crystallization

-Lysozyme batch cooling crystallization in the presence of two ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), and 1,3-dimethylimidazolium iodide ([C1mim]I). Microscopic observation demonstrated that the crystal morphology with [C1mim]I is needle-like, while that with [C4mim]Cl and no ILs is plate-like. FTIR spectroscopy revealed that new peaks in the range of 1300–400 cm−1 occur with the case of adding [C1mim]I, meaning that the microstructure of lysozyme crystals changed. Raman spectroscopy and X-ray powder diffraction (XRD) pattern further confirmed that crystal forms of lysozyme with adding [C4mim]Cl and no ILs is completely different from that of adding [C1mim]I. Thermogravimetry (TG) and Differential Scanning Calorimeter (DSC) results indicated that addition of [C1mim]I delay the thermal deformation and degradation temperature of lysozyme crystals, suggesting that lysozyme crystals with [C1mim]I possesses higher thermal stability. The change in lysozyme crystal forms can be attributed to changes in interaction among lysozyme molecules and conformation in solution, leading to different assembly mode of molecules and formation of polymorphs. These findings could offer instructions for regulating lysozyme crystal forms and gaining initial insight into screening of potential ILs for developing polymorphs of the protein and other macromolecule crystals.

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.

Droplet microfluidics for time-resolved serial crystallography.

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.

Data‐driven identification of crystallization kinetics

AbstractA novel data‐driven methodology is presented for developing mathematical models for crystallization processes. The data‐driven approach is based on the sparse identification of nonlinear dynamics (SINDy) method, which iterates between a partial least‐squares fit and a sparsity‐promoting step leading to the discovery of sparse interpretable models. The performance of the SINDy methodology is characterized for the identification of crystallization kinetics in a mixed tank operated in a continuous mode, the isothermal crystallization of lysozyme in a batch stirred tank and cooling crystallization of paracetamol. The SINDy method is robust against noise. Good agreement is obtained between the data‐driven model and the data obtained from crystallization experiments. The presented data‐driven approach can be attractive for modeling industrial crystallization processes where process analytical technology tools are available for the measurement of process variables but functional forms of kinetic expressions are unknown.

PEGDA hydrogel microspheres with encapsulated salt for versatile control of protein crystallization

Due to their biocompatibility and adjustable chemical structure and morphology, hydrogels have great potential in many applications, and can be used to enhance protein crystal quality and crystallization efficiency, contributing to biomedicine manufacturing. Monodispersed PEGDA hydrogel microspheres (HMSs) were synthesized using a Lego-inspired microfluidic device. The generated droplets were then UV polymerized, partially hydrolyzed with 0.1 M NaOH solution to improve their absorption capacity, and soaked in a buffer solution containing 0, 0.5, 1, 2, and 4 M NaCl. Salt-loaded HMSs were used as the medium for the enhanced crystallization of hen egg white lysozyme from aqueous solutions. Different supersaturations were achieved in the protein solutions by releasing NaCl of different concentrations from HMSs, as confirmed by electrical conductivity measurements. HMSs with or without NaCl can both provide heterogeneous nucleation sites due to their nano-porous structure and wrinkled surface. The addition of NaCl-loaded HMSs to the protein solution can also increase or decrease the supersaturation in the whole solution or locally near the HMS, leading to controllable nucleation time and crystal size distribution dependent on the NaCl concentration loaded into HMSs.

The Modification of Dissolution Kinetics and Solubility of Lysozyme Crystals by Ionic Liquids

The Modification of Dissolution Kinetics and Solubility of Lysozyme Crystals by Ionic Liquids

Surfactant-assisted crystallization of lysozyme and its mechanism

Surfactants were used to assist in the crystallization of lysozyme. Three types of surfactants with different structures (sodium dodecyl sulfate, cetyltrimethylammonium bromide, or betaine hydrochloride [BH]) were used. The study indicated that three surfactants were all capable of shortening the crystallization induction time of lysozyme, with BH inducing the shortest crystallization induction time (t = 1 min) and the largest crystal size (37 μm). The molecular interactions among each of the three surfactants aforementioned and lysozyme were also investigated by simulations. It was found that hydrogen bonding dominantly acted between the carboxyl structure of the surfactant BH and the lysozyme, which produced the most favorable conformation for the self-assembly of lysozyme (promote lysozyme orderly aggregation and shorten the crystallization induction time). On this basis, the mechanism of surfactant-promoted lysozyme crystallization was proposed.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

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

High-Resolution Electron Diffraction of Hydrated Protein Crystals at Room Temperature.

Structural characterization is crucial to understanding protein function. Compared with X-ray diffraction methods, electron crystallography can be performed on nanometer-sized crystals and can provide additional information from the resulting Coulomb potential map. Whereas electron crystallography has successfully resolved three-dimensional structures of vitrified protein crystals, its widespread use as a structural biology tool has been limited. One main reason is the fragility of such crystals. Protein crystals can be easily damaged by mechanical stress, change in temperature, or buffer conditions as well as by electron irradiation. This work demonstrates a methodology to preserve these nanocrystals in their natural environment at room temperature for electron diffraction experiments as an alternative to existing cryogenic techniques. Lysozyme crystals in their crystallization solution are hermetically sealed via graphene-coated grids, and their radiation damage is minimized by employing a low-dose data collection strategy in combination with a hybrid-pixel direct electron detector. Diffraction patterns with reflections of up to 3 Å are obtained and successfully indexed using a template-matching algorithm. These results demonstrate the feasibility of in situ protein electron diffraction. The method described will also be applicable to structural studies of hydrated nanocrystals important in many research and technological developments.

The regulation of protein crystallization using choline chloride precipitant and its mechanism

Inorganic salts are the most commonly used precipitants in protein crystallization. However, due to their strong ability to destroy the hydration shell and diffuse electric double layer of proteins, the use of inorganic salts can easily cause protein precipitation. This work studied protein crystallization using organic salt choline chloride (cc) as the precipitant. Compared with NaCl, crystallization, it was found that lysozyme could nucleate at a lower supersaturation and the single-crystal area was significantly enlarged with cc. Even at a high supersaturation of nearly 30, lysozyme crystals could still maintain a complete tetragonal morphology with no urchin-like crystals in cc solution. Furthermore, the average crystal size in cc solution was larger than that in NaCl solution even at a lower supersaturation. In addition, the quantitative efficiency of yield was about equal in cc and NaCl system. Combining Zeta potential, fluorescence spectrum and MD simulation, it was found that cc could interact with lysozyme through hydrogen bond, which weakened the damage of Cl− to diffuse electric double layer of proteins and increased the stability of lysozyme. DLS revealed that the presence of cc could promote the formation of large protein aggregates, which was favorable for crystal growth. In addition, CD spectra and lysozyme activity showed that the presence of cc did not affect the secondary structure and activity of lysozyme. This work proved that cc could be used as a substitute precipitant to grow protein crystals with large size and improve the stability of protein crystallization.

The changes in molecular interaction and conformation of lysozyme mediated by ionic liquids

Using Ionic liquids（ILs）to regulate protein crystallization has attracted great attention, but the mechanism of ILs mediating crystallization is still unclear. Herein, we chose 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and 1,3-dimethylimidazolium iodide ([C1mim]I) as additives to mediate lysozyme crystallization, and use situ Raman to track the conformational changes of lysozyme in solution. Meanwhile, molecular simulation was used to explore the binding between ILs and lysozyme molecules. The results demonstrated that the addition of ILs produced new characteristic peaks in Raman spectra, suggesting some changes to occur in solution structure. The changes of peaks at 1340 cm−1 and 1359 cm−1, and the enhancement of peak at 1450 cm−1 indicated the unfolding of lysozyme molecules. The quantitative analysis of amide Ⅰ band showed that the addition of ILs increased the β-sheet content in secondary structures. Molecular docking showed that [C4mim]Cl and [C1mim]I could both bind to the active site of lysozyme molecules, and the binding energy were −3.1 kcal/mol and −4.5 kcal/mol, respectively. Molecular dynamics results showed that [C4mim]Cl could stably bind to lysozyme molecules, while [C1mim]I left the active site after several ns, which means that [C4mim]Cl brought about stronger impact on interaction of lysozyme molecules than [C1mim]I in solution.

Optimization of Batch Crystallization of Magnetic Lysozyme Crystals and Study of the Continuous Crystallization Process

Protein crystallization is a widely employed technique for purifying protein drugs, offering notable benefits such as cost-effectiveness and high purity. However, the success of this method is influenced by factors such as the molecular weight and spatial structure of proteins. The challenges associated with achieving crystallization and the prolonged duration required for crystallization induction pose limitations on its widespread industrial implementation. In this study, we employed lysozyme derived from egg white as a representative protein to investigate the polymer-assisted self-assembly of magnetic lysozyme. Through the optimization of the initial interstitial crystallization process of magnetic lysozyme, we manipulated the supersaturation level of lysozyme and applied magnetic nanoparticle treatment. As a result, we successfully reduced the crystallization time from 24 h to 60 min. Subsequently, the findings derived from the analysis of data pertaining to the interstitial crystallization process of lysozyme were utilized to optimize the design and configuration of a push flow crystallizer (PFC) as well as a slug flow crystallizer (SFC). The analysis encompassed the examination of various factors, including the residence time of crystallization, the yield of the process, the shape of the crystals formed, and the distribution of crystal sizes. Ultimately, it was determined that the SFC demonstrated optimal suitability for the crystallization of magnetic lysozyme. The typical V-PFC crystal size is 16 m and the yield is 60%. V-SFC crystals have an average size of 13 m and a yield of 85%.

Optimization on crystallization process of the magnetic core lysozyme

The continuous growth of the biomedical market highlights the significance of improvement in the efficiency of batch production for protein drug crystals. Herein, we investigated the effect of polymer-modified Fe3O4 nanoparticles on lysozyme (Lys) crystallization at different conditions, using functionalized Fe3O4 magnetic nanoparticles (Polymer C@Fe3O4) as nucleation agents. The study revealed that the surface charge density of Polymer C@Fe3O4 was highest at a modification mass ratio of Fe3O4 to Polymer C is 8:3. This ratio facilitates the augmentation of electrostatic interaction and the promotion of crystallization. By adding 15% of the PolymerC@Fe3O4 prepared from Fe3O4 with an average particle size of 150 nm, the magnetic core lysozyme (M-Lys) crystals with an average particle size of 11.08 μm, narrow size distribution, and regular morphology were obtained in a yield of 82.42% within 60 min.

Protein crystallization with DNA templates

Protein crystallization plays a significant role in three-dimensional structural analysis and protein purification. It is important to increase the crystallization efficiency, which is possible by adding heterogeneous templates in crystallization systems. DNA is biologically compatible and artificially designable polymer, which is easy to extract. In this study, single- and double-stranded DNA of precise sequences were designed and used as templates to promote protein crystallization of lysozyme and catalase. Influence of DNA, single-stranded DNA with 10, 20, 40 bases and double-stranded DNA with 10, 20, 40 base pairs, were investigated. The success rate of obtaining crystals of lysozyme and catalase in equal period was significantly improved with the addition of DNA comparing without templates added. Double-stranded DNA led to higher nucleation rate than that with single-stranded DNA. The promotion of nucleation was more obvious at low concentration of protein solution and with longer chain DNA templates. Crystal number and crystallization rate was enhanced with addition of long double-stranded DNA templates. All the results confirm that DNA is an effective polymer additive to enhance protein crystallization, especially for the application of the scarce protein crystallization.

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.