Orthorhombic Lysozyme Crystals Research Articles

Theory of the Solubility of Protein Crystals

We present a theory describing the solubility of protein crystals as a function of pH, salt concentration, and temperature. There are four terms in the model. The neutral terms arise from 1) the translational entropy of the soluble proteins, and 2) H-bond and hydrophobic attractive interactions which we obtain from a fitting procedure. The two electrostatic terms are a result of counterions confined in the crystal to satisfy charge neutrality. These counterions contribute 3) an entropic penalty from the trapping of ions in the crystal, and 4) a favorable enthalpy from the interaction of each protein with its counterion cloud. This theory quantitatively describes the solubility of tetragonal and orthorhombic lysozyme crystals as determined by Pusey et al. According to the theory, the reduced solubility at high salt concentrations comes, not from increased screening, but from a reduced entropy of counterion confinement. The theory correctly describes the weak pH dependence of the solubility, which is a result of the compensating effects of the two electrostatic terms. We discuss the implications of this theory for crystal nucleation and the success of the “crystallization slot”.

Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue

AbstractThe reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach‐Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Shape of steps on the (010) face of orthorhombic lysozyme crystals

An attempt is made to explain the shape of the curved steps at the (010) face of orthorhombic lysozyme crystals and to determine the density, the rate of formation, and the velocity of kinks. The interpretation is performed in terms of the concepts developed in studies on the crystallization of long-chain polymers within the Frank model. It is demonstrated that this model is incorrectly generalized to the case of high kink densities. A algorithm for determining the coordinates of the growth front in images obtained with atomicforce microscopes is proposed and implemented.

Formation of protein crystals (orthorhombic lysozyme) in quasi-microgravity environment obtained by superconducting magnet

As one of the best candidates for simulating the microgravity conditions in space, low gravity environments provided by applying an upward magnetic force have been considered to grow protein crystals. Since 2002, the stable and long-time durable microgravity generated by a superconducting magnet has been available for protein crystal growth. In this paper, for the first time, we grew protein crystals (orthorhombic lysozyme crystals) at quasi-microgravity. The present study showed that quasi-microgravity improves the crystal quality effectively and reproducibly. The application of strong magnetic field also improves the crystal quality. Furthermore, it is possible to know if microgravity is effective for the improvement of crystal quality or not by growing crystals in microgravity and hypergravity inside the superconducting magnet and comparing the crystal quality with each other. Such test will be useful to select promising proteins prior to the space experiments.

Significant Effects of Magnetic and Gravitational Fields on the Morphology of Protein Crystals (Orthorhombic Lysozyme Crystals Grown Using NiCl2 as Crystallization Agent)

Magnetic fields (either horizontal or vertical) were found to drastically change the morphology of protein (orthorhombic lysozyme) crystals. A pair of {011} faces evident in crystal grown in the ab...

Effects of high pressure on the growth kinetics of orthorhombic lysozyme crystals

Growth processes of the (0 1 1) and (0 1 ̄ 1) faces of an orthorhombic lysozyme crystal were observed in situ under 0.1 and 100 MPa. The growth rates of the (0 1 1) and (0 1 ̄ 1) faces were equal within an experimental error. From the comparison between the growth rates under 0.1 and 100 MPa at the same supersaturation level, we found that the growth kinetics of the orthorhombic lysozyme crystals significantly decreases with an increase in pressure. Supersaturation dependencies of the growth rates under 0.1 and 100 MPa were well fitted with a two-dimensional (2D) nucleation growth model of a birth-and-spread type. The fitting results show that the decrease in the growth kinetics with pressure can be explained by the increase activation energies in the incorporation processes of solute molecules.

Dependence of the step velocity on its length on the (010) face of the orthorhombic lysozyme crystal

The length of a two-dimensional critical nucleus has been measured, and the Gibbs-Thomson formula has been experimentally verified on orthorhombic lysozyme crystals. The step velocity was found to be independent of its length. The critical length initiating the step motion can be determined by a low density of kinks on a step, and not the critical-nucleus size. The invalidity of the Gibbs-Thomson formula in this case is discussed.

Improvement in diffraction maxima in orthorhombic HEWL crystal grown under high magnetic field

Orthorhombic crystals of hen egg-white lysozyme (HEWL) were grown under a homogeneous magnetic field of 10 T ( B=10 T). In the magnetic field, crystals were oriented such that their crystallographic c-axes were parallel to the magnetic field, and gave narrower average rocking widths than those grown under 0 T. Crystal quality was evaluated using the full-width at half-maximum (FWHM) of the rocking curve, and the effects of a magnetic field were examined by comparing FWHM of seven crystals grown at 10 T with an equal number grown at 0 T. FWHM values in (10 0 0), (0 8 0), and (0 0 6) reflections decreased by 25%, 38%, and 32%, respectively, and those of general reflections decreased by 7–45% in crystals grown under high magnetic field. These results show that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. At the same time, the maximum resolution limit of X-ray diffraction increased from 1.33 Å for 0 T to 1.13 Å for 10 T. These facts suggest that the application of magnetic field for crystallization might generally have a striking effect in enhancing the diffraction power of protein crystals.

Enhancement in the perfection of orthorhombic lysozyme crystals grown in a high magnetic field (10 T)

Orthorhombic crystals of hen egg-white (HEW) lysozyme were grown in a homogeneous and static magnetic field of 10 T. All crystals grown at 10 T were oriented such that their crystallographic c axes were parallel to the magnetic field direction and showed a narrower average full-width at half-maximum (FWHM) of the rocking curve than those grown at 0 T. Rocking-width measurements were made at the BL-10A station at the Photon Factory, Tsukuba, Japan, using a high-resolution vertical-type four-circle diffractometer. Crystal perfection was evaluated using the FWHM of the rocking curve; the effects of the magnetic field on the quality of the crystals were examined by comparison of the FWHM of seven crystals grown at 10 and 0 T. The FWHMs of the reflections along the a, b and c axes decreased by 23.5, 35.3 and 27.8%, respectively, and those of other general reflections decreased by 17.4-42.2% in the crystals grown at high magnetic field. These results clearly showed that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. As a result, the maximum resolution of X-ray diffraction increased from 1.3 A at 0 T to 1.13 A at 10 T. The magnetic field also affected the dimensions of the unit cell, increments being 0.2% for the a and c axes and 0.1% for the b axis, respectively. These facts suggest that the application of a high magnetic field during crystallization might result in remarkable enhancements in the diffraction power of protein crystals having magnetic anisotropy.