Growing large-volume protein crystals bigger, better in space

A group of researchers from the University of Alabama in Huntsville, iXpressGenes, University of Grenada in Spain, and Oak Ridge National Laboratory designed microgravity experiments to grow crystals of inorganic pyrophosphatase (IPPase) in space. IPPase is an enzyme found in most living organisms that plays an important role in bone formation, DNA synthesis, and the making and breaking down of fats. The researchers' goal was to grow high-quality, large-volume crystals for use in neutron macromolecular crystallography (NMC), which is the preferred method for determining the positions of hydrogen atoms within macromolecules.

The group will present their findings during the American Crystallographic Association's 66th Annual Meeting, in Denver, Colorado, July 22-26.

"Although hydrogen constitutes 50 percent of the atoms in proteins of the 100,000 plus X-ray structures reported in the RCSB Protein Data Bank, NMC has been used to identify fewer than 100 unique protein structures," said Joseph D. Ng, director of the Biotechnology Science & Engineering Program, Department of Biological Sciences, University of Alabama in Huntsville. "The major factor limiting the use of NMC is the inability to obtain the large crystal volumes (~1 cubic millimeter) necessary for neutron diffraction data."

The group launched their project by first making test crystals of IPPase on the ground at the Grenada Crystallization Facility.

The crystallization system they developed uses capillary tubes to control the diffusion rate of precipitating chemical reagents against dissolved protein molecules in a solution. The geometry forces the molecules to concentrate in part of the solution, which then becomes supersaturated, meaning there are too many molecules to stay comfortably dissolved. The molecules then come out of solution to form a crystal.

The system was designed to work best under microgravity, since the forces of gravity can affect the flow of the solution. It was sent into space via SpaceX, for crystallization aboard the International Space Station (ISS), where "proteins can crystallize in an optimized supersaturated condition," said Ng.

Remarkably, the project's hardware launched into space inside a cargo transfer bag at ambient temperature and didn't require any ISS crew interaction.

"Two GCF units were launched on SpaceX-3, and then returned six months later on SpaceX-4," Ng said. "From these flights, IPPase crystals with volumes greater than 6mm3 were obtained in 2-mm quartz capillaries." To date, the largest known IPPase crystals were obtained from these experiments aboard the ISS.

How do space-grown crystals compare to Earth-grown ones? For the majority of comparisons, space-grown crystals were "superior," Ng said.

Diffraction analyses from both neutron and X-ray sources showed structures "with higher resolution and precision than their Earth equivalents," Ng added. "In particular, the large-volume crystals provided neutron diffraction information that revealed hydrogen locations that couldn't have been determined using other methods. In other words: the determined structures from space-grown crystals are valuable therapeutic targets for initial drug modeling."

This may help to ease a drug design bottleneck that was caused by "the lack of crystallization successes for protein molecules of pharmaceutical interest that can lead to high-resolution structures suitable for drug modeling," said Ng. "But now, we see the promise of obtaining well-formed, large-volume crystals grown in space via the ISS platform to help overcome the rate-limiting step involved in the drug modeling process."

If large crystals can be obtained, a structure determined by NMC can be effectively applied to develop a drug delivery solution -- and may provide opportunities for the pharmaceutical industry to increase speed or reduce costs.

"Microgravity crystal growth for NMC -- to produce accurate atomic locations of pharmaceutical targets -- could become a major application for the space station," Ng said.