High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors.

Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D(2), an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC(50)) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC(50) value of 50 nM extended to 1.1 A resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.