Macromolecular crystallization in microgravity generated by a superconducting magnet.

About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

Strong magnetic field effect on the dissolution process of tetragonal lysozyme crystals.

Either a homogeneous or inhomogeneous magnetic field has been known to dampen the protein crystal growth. To date the mechanism is not clear. However, it was generally proposed that the magnetic field may dampen the convection in the solution, resulting in a reduced crystal growth rate and possibly a good crystal quality, similar to the case of protein crystal growth in space. To understand the mechanism of the magnetic field effect on protein crystal growth, further explorations on the magnetic field effect on protein solution, on the processes of crystal growth and dissolution, and on different crystallization (solution) systems, should be valuable. In this paper we present our recent efforts to study magnetic field effects on the dissolution processes of tetragonal lysozyme crystals under a strong magnetic field. A layer of oriented tetragonal lysozyme crystals was prepared under a temperature gradient and magnetic field, after that the crystals were dissolved by increasing the temperature of the solution. The lysozyme molecules will diffuse upwards due to the steep concentration gradient at the lower side of the cell caused by the dissolution. The evolution of the concentration in the solution was measured in-situ using a Mach-Zehnder interferometer. The results confirmed that the dissolution process of the crystals was slowed by the magnetic field. Judging from the concentration evolution versus time at different positions in the solution, we concluded that the apparent diffusion coefficient of lysozyme molecules was decreased by the magnetic field. The results were discussed using a suspended crystal model in the initial dissolution stage.

An investigation of magnetic field effects on the dissolution of lysozyme crystal and related phenomena.

It is now widely known that a magnetic field, either homogeneous or inhomogeneous, depresses the growth process of protein crystals. In this report, the dissolution process of tetragonal lysozyme crystals is also confirmed to be depressed by a homogeneous magnetic field (inhomogeneity <1.5%). The dissolution process was monitored using a Mach-Zehnder interferometer. The results showed that the concentration change during the dissolution process was slowed in a magnetic field compared with that in the absence of a magnetic field. It was concluded that the diffusion coefficient of the lysozyme molecules in the solution was decreased by the magnetic field. The decrease in the diffusion coefficient may contribute to the slowed growth process. The changes in the spatial concentration distribution under a vertical temperature gradient before crystallization in the absence of a magnetic field was also studied. The concentration in the lower, colder part of the cell increased, while it decreased in the upper, hotter part, a similar phenomenon to that discovered by previous investigators in an isothermal supersaturated solution system. Aggregated domain formation is proposed to explain the concentration redistribution before crystal growth and a suspended crystal model is proposed to explain the decrease of diffusivity in a magnetic field.

Magnet used for protein crystallization: novel attempts to improve the crystal quality.

The accuracy of the structures of biological macromolecules determined by X-ray crystallography is of fundamental importance, both for the understanding of life processes and for medical applications. The resolution of the structure is thus critical, and is largely determined by the quality of single crystals. Here we report the results of applying a magnetic field and a magnetization force during growth of the snake muscle fructose-1,6-bisphosphatase and human estrogenic 17beta-hydroxysteroid dehydrogenase crystals. For both enzyme proteins, the quality of the crystals improved with repeated assay, and their data sets were collected at significantly higher resolutions. These results coincide with a mechanism involving the reduction of convection, due to both the hydrodynamics within a magnet and the partially reduced gravity induced by a magnetization force. The density difference between the crystal and solution becomes less significant, and the sedimentation speed of the crystals is also reduced in the presence of the magnetization force.