Mitsuo Ataka

Effect of a magnetic field gradient on the crystallization of hen lysozyme

The effect of microgravity on protein crystal growth is an interesting, but highly controversial, issue. We superimposed the magnetic force on gravity and studied its effect on the crystal growth of hen egg-white lysozyme. The magnetic force was used similarly to the centrifugal force in a space shuttle to virtually change the level of gravity. We observed that the number of crystals increased or decreased, respectively, when 5% of the additional force was applied in the same or in the opposite direction to normal gravity. Our result indicates that the magnitude of gravity can in fact affect the studied protein crystal growth, and that a magnetic force has the potential ability of controlling gravity.

Systematic studies on the crystallization of lysozyme: Determination and use of phase diagrams

Abstract The concentration changes which accompanied the crystal growth of hen egg-white lysozyme from its aqueous solution were followed optically at 5, 10, 15, 30, 42, and 50°C for 30–360 days in the presence of 1–7% NaCl. When the initial protein concentration was selected so as to start the crystallization within a reasonable time and with a proper number of nuclei, the supernatant concentration approached constant values after 1–1.5 month, which were regarded as solubility ( S ) of the crystals. Phase diagrams representing S as functions of NaCl content and temperature were obtained for two crystal forms of lysozyme. From the Arrhenius plot, the enthalpy of crystallization for the tetragonal form was calculated to be − 72 kJ/mol, and that for the orthorhombic form to be −32 kJ/mol. Conditions necessary for obtaining accurate phase diagrams were discussed, together with their possible use for crystal growth and for future molecular engineering.

Magnetic orientation as a tool to study the initial stage of crystallization of lysozyme

Abstract The tetragonal crystals of hen egg-white lysozyme align their c -axis in the direction of a magnetic field. By applying the magnetic field of 1.6 T only over some period during the whole crystallization process, it was possible to know when the crystals sedimented. It was found that crystals grew in solution, and started to sediment on reaching a critical size. We evaluated the critical size to be 1–2 μm by changing the magnetic field strength (0.1–1.2 T) and analyzing the relation between the field strength and the proportion of magnetic orientation.

Low-Frequency Raman Spectra of Lysozyme Crystals and Oriented DNA Films: Dynamics of Crystal Water

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.

Laser Michelson interferometry investigation of protein crystal growth

Laser Michelson interferometry was applied to study the elementary growth mechanism of protein crystals. The results for the (101) face of tetragonal lysozyme show that for supersaturations σ higher than 1.6, growth proceeds by two-dimensional nucleation. However, at lower supersaturations growth is governed by dislocation sources. The observed non-linearity of the step velocity versus supersaturation dependence for supersaturations up to 1.2 is proved to be due to strong impurity effects. At σ < 0.4 the crystal surface is covered with macrosteps. The effective step kinetic coefficient for the studied face is determined: β = 2.8 × 10−6 m/s. The applicability of general crystal growth principles and theories to protein crystallization is thus illustrated.

Aggregation in supersaturated lysozyme solution studied by time-resolved small angle neutron scattering

Abstract A model of the lysozyme crystallization process has been given by time-resolved small angle neutron scattering from lysozyme in supersaturated solution. In supersaturated solution larger aggregates (Type I), with a radius greater than several hundred angstrom, co-exist with a much larger number of smaller particles (Type II), the radius of which is between 25 and 40 A. The size distribution of Type I is poly-dispersive and that of Type II is monodispersive. As time elapses, the structure of Type I aggregates does not change so much. On the other hand, the radius of Type II aggregates increases with time, but stops at about 14 h, and then decreases. Nucleation occurs at about 14 h.

Protein crystallization induced by polyethylene glycol: A model study using apoferritin

The phase behavior of apoferritin solutions induced by the addition of polyethylene glycol (PEG) was studied. The interaction between apoferritin molecules was determined by dynamic light scattering. The comparison of the experiments with the theoretical calculations showed that the addition of NaCl to the protein solution only screened the electrostatic repulsion and did not induce attraction. By the addition of PEG, on the other hand, significant attraction was induced and three types of precipitation (crystals, liquid domains, and random aggregates) appeared depending on the concentration of PEG and on its molecular weight. The strength of the attraction could be explained by the depletion mechanism, although there was slight discrepancy between the simple theory and the experiments. Superiority of PEG is thus demonstrated since the depletion mechanism does not depend on specific nature of proteins. From the phase diagram, we suggest that the control of the concentration and molecular weight of PEG are...

Kinetic studies on the growth of three crystal forms of lysozyme based on the measurement of protein and Cl- concentration changes

Abstract The kinetics of hen egg-white lysozyme crystallization is measured as a function of pH at 5 and 35°C, using batch experiments. Conditions are formulated for which a theory of protein self-assembly can be applied to the crystallization of lysozyme. Furthermore, the influence of low-molecular-weight ions upon crystallization is analyzed.

Magnetic suppression of convection in protein crystal growth processes

Magnetization force caused by a magnetic field gradient (F m ) is a body force and can cause buoyancy. We numerically simulate the natural convection arising from the depletion of protein concentration around a growing protein crystal when an upward magnetization force acts on the solution. The numerical predictions reveal that an upward magnetization force can damp convection. When a magnetic field gradient μ 2 0 H(dH/dz) = -685 T 2 /m is applied, the maximum flow velocity is reduced by about 50% and the velocity in the vicinity of the crystal is reduced by about 24%. Due to the low electric conductivity of the solution, the contribution of the Lorentz force is negligible. When μ 2 0 H(dH/dz) = -1370 T 2 /m, the upward magnetization force (F m ρg) damps convection completely. Our study shows a new method of controlling convection in the process of protein crystal formation.

Orientation of protein crystals grown in a magnetic field

Abstract Three crystal forms of hen egg-white lysozyme, and ribonuclease A and met-myoglobin crystals exhibited orientation in a magnetic field of