Tetragonal Hen Egg-white Lysozyme Crystals Research Articles

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.

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.

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.

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.

Crystallization of high-quality protein crystals using an external electric field

The effect of a 20 kHz external electric field on the quality of tetragonal hen egg white (HEW) lysozyme crystals was investigated using X-ray diffraction rocking-curve measurements. The full width at half-maximum was found to be larger for high-order reflections but smaller for low-order reflections. In particular, it was revealed that a large amount of local strain is accumulated in tetragonal HEW lysozyme crystals grown under an applied field at 20 kHz. Comparison with previous results obtained for crystals grown with an applied field at 1 MHz [Koizumi, Uda, Fujiwara, Tachibana, Kojima & Nozawa (2013).J. Appl. Cryst.46, 25–29] indicated that improvement of the protein crystal quality could be achieved by selection of an appropriate frequency for the applied electric field, which has a significant effect on the growth of the solid.

First Direct Observation of Impurity Effects on the Growth Rate of Tetragonal Lysozyme Crystals under Microgravity as Measured by Interferometry

The normal growth rates R and apparent step velocities (lateral growth rates of a spiral hillock) V of tetragonal hen egg-white lysozyme (HEWL) crystals were for the first time measured by Michelson interferometry in the international space station (as part of the NanoStep project) using commercialized HEWL samples containing 1.5% impurities. A significant increase in V under microgravity was confirmed compared to step velocities Vstep on the ground, while a decrease in R was also confirmed compared to that in the purified solution under microgravity as expected. Because of exact measurement of growth rates, kinetic analyses of R were conducted as a function of supersaturation, σ (σ ≡ ln(C/Ce), where C is the concentration; Ce is the solubility), using a spiral growth model and a two-dimensional (2D) nucleation growth model. For both models over a wide range of σ, R in the impure solution was significantly lower than that in the purified solution. The degree of the suppression of impurity effects was also...

Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station.

The normal growth rates of the {110} faces of tetragonal hen egg-white lysozyme crystals, R, were measured as a function of the supersaturation σ parameter using a reflection type interferometer under μG at the International Space Station (NanoStep Project). Since water slightly evaporated from in situ observation cells during a long-term space station experiment for several months, equilibrium temperature T(e) changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration C(s). To correct σ, the actual C(s) and protein concentration C(p), which correctly represent the measured T(e) value in space, were first calculated. Second, a new solubility curve with the corrected C(s) was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the C(s) and C(p) of the solution.

Control of Subgrain Formation in Protein Crystals by the Application of an External Electric Field

X-ray diffraction rocking-curve measurements were performed on tetragonal hen egg white (HEW) lysozyme crystals grown with and without the application of an external alternating current (AC) electric field. Analysis of the full width at half-maximum (fwhm) of the rocking curves indicated that misorientation between subgrains, |θ – φ|, and local strain, ⟨e⟩, in the crystals contributed to the broadening of the experimentally determined rocking curves for the tetragonal HEW lysozyme crystals. In particular, the data suggested that imperfections in tetragonal HEW lysozyme crystals are predominantly caused by misorientation between subgrains. Moreover, it was determined that improvements in the quality of tetragonal HEW lysozyme crystals, as assessed by a decrease in the misorientation between subgrains, can be achieved by imposing a 1 MHz electric field during crystal growth. This paper also discusses the origin of subgrain misorientation in light of the incorporation of impurities into protein crystals.

Digital topography with an X-ray CCD camera for characterizing perfection in protein crystals

Digital X-ray topography using an X-ray CCD camera and conventional X-ray topography using X-ray film were used to investigate tetragonal hen egg-white lysozyme (HEWL) crystals. Previously, clear dislocation images of protein crystals were mainly obtained by film methods. Earlier studies of HEWL crystals using an X-ray CCD camera mainly revealed domain structures. In the present study, dislocation images of the same HEWL crystal have been obtained by using conventional X-ray film and a digital X-ray CCD camera. The results demonstrate that digital topography using an X-ray CCD camera is an effective method for characterizing protein crystals. A series of digital topographic images were analyzed by the method developed by Lovelace, Murphy, Pahl, Brister & Borgstahl [J. Appl. Cryst.(2006),39, 425–432]. Sub-peaks and peak broadening originating from dislocations in local rocking curves were observed. Moreover, the crystal perfection was evaluated by mapping the angular positions of the maximums and the full widths at half-maximum of local rocking curves.

Solubility measurements by in situ observation of the apex region formed by the (110), (11̄0) and (101) faces of tetragonal lysozyme crystals

Equilibrium temperatures, Te, of tetragonal hen egg-white lysozyme crystals were determined by in situ observation of apex regions formed by the (110), (11̄0) and (101) faces of the crystals. We could precisely measure Te within 60min, and with an error margin ±0.7°C or less. This is largely because growth and dissolution can be detected more rapidly at the apex than at the steps at the center of the crystal faces.

Effect of various precipitants on the nucleation rate of tetragonal hen egg-white lysozyme crystals in an AC external electric field

It was observed that the effect of an external electric field on the nucleation rate of tetragonal hen egg-white lysozyme crystals varied depending on the precipitant used (NaCl, NiCl 2 or YbCl 3) and that the electric double layer (EDL) played an important role in generating an external electric field of the necessary strength to control the nucleation rate. This phenomenon depended on the ionic strength of the precipitant used; that is, a precipitant of greater ionic strength resulted in a thinner EDL and increased the effect of the external electric field as the driving force for nucleation. The dependence of the nucleation rate on the precipitant was attributed to the magnitudes of the external electric fields generated in EDLs of varying thickness which were formed in the presence of different precipitants.

Macromolecular crystallography at high pressure with pneumatic diamond anvil cells handled by a six-axis robotic arm

A new pneumatic diamond anvil cell has been constructed, generating continuous pressure and temperature variations in the range 0–2.5 GPa and 293–393 K. The cell is designed mainly for high-pressure macromolecular crystallography and should facilitate pressure and temperature annealing of the sample. The analysis is reported of several diffraction data sets of tetragonal hen egg-white lysozyme crystals loaded either in the new cell or in a currently used membrane-based cell. These experiments were performed on beamline FIP-BM30A at the ESRF, Grenoble, a macromolecular crystallography beamline on a bending magnet. Cells were handled and automatically centred by a six-axis robotic arm that was used as a goniometer for data collection by the oscillation method.

Solubility measurements of protein crystals under high pressure by in situ observation of steps on crystal surfaces

We successfully measured equilibrium temperatures Te of tetragonal hen egg-white lysozyme crystals under high pressure by in situ observation of steps on the {110} faces of the crystals. The dependence of Te on the concentration of lysozyme corresponds to that of the solubility Ce on temperature. The precision of the solubility determined in this study is significantly higher than that in previous works. One Te could be measured during short time less than 70 minutes. This method for solubility measurements of protein crystals under high pressure is the fastest one at this stage.

Phase transition in tetragonal hen egg-white lysozyme crystals

Lattice dynamics of tetragonal hen egg-white lysozyme crystals was studied by Brillouin light scattering and differential ac-nanocalorimetry with controlled relative humidity from 298 to 330 K. Temperature dependences of fast quasitransverse phonon velocity and integral intensity were found to exhibit anomalies in the vicinity of 306 K. An anomaly in the temperature behavior of heat capacity was also observed near this temperature. It is concluded that the anomalies point to a structural phase transition. Possible mechanisms of phase transition in lysozyme crystals are discussed.

Elastic constants in tetragonal hen egg-white lysozyme crystals containing large amount of water

Transverse sound velocity of cross-linked tetragonal hen egg-white (HEW) lysozyme crystals containing large amount of water in the crystal was measured using ultrasonic pulse-echo method. All elastic constants of cross-linked crystals were observed to be C11=C22=5.50 GPa, C12=4.33 GPa, C13=C23=3.94 GPa, C33=5.22 GPa, C44=C55=0.68 GPa, and C66=0.84 GPa, respectively. We found that the elastic constants of the cross-linked crystals are identical to those of the intrinsic ones without cross-linking. Moreover, we found that tetragonal HEW lysozyme crystals that enclose large amount of water show decreased elastic constants (softening). In particular, the shear elastic constants C44=C55 and C66 showed more softening effect comparing with other elastic components.

Control of Nucleation Rate for Tetragonal Hen-Egg White Lysozyme Crystals by Application of an Electric Field with Variable Frequencies

Control of the nucleation rate is important for the growth of high quality protein crystals; therefore, the development of improved growth methods is required. Crystal growth with an external electric field is an attractive method for control of the nucleation rate. However, mainly a decrease in nucleation rate was observed when an external electric field was applied using DC voltage. By focusing on the chemical potential of the liquid and solid, control of both the increase and the decrease in nucleation rate was successfully achieved by applying an AC electric field. Control of nucleation rate in terms of enhancement and retardation is reported for hen-egg white lysozyme crystals by imposition of an electric field with varying frequencies, which was determined by the electrical permittivity of both the hen-egg white lysozyme solution and crystals.