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

Inaka K, Takahashi S, Aritake K, Tsurumura T, Furubayashi N, Yan B, Hirota E, Sano S, Sato M, Kobayashi T, Yoshimura Y, Tanaka H, Urade Y - Cryst Growth Des (2011)

Bottom Line: 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.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.The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0).

View Article: PubMed Central - PubMed

Affiliation: Maruwa Foods and Biosciences Inc. , 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

(a) Crystals of wild-type L-PGDS grown in microgravity. (b) Crystals of the C65A mutant grown in microgravity. (c) Crystals of wild-type L-PGDS grown on the ground. (d) Crystals of the C65A mutant grown on the ground. The scale bars correspond to 100 μm.
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fig2: (a) Crystals of wild-type L-PGDS grown in microgravity. (b) Crystals of the C65A mutant grown in microgravity. (c) Crystals of wild-type L-PGDS grown on the ground. (d) Crystals of the C65A mutant grown on the ground. The scale bars correspond to 100 μm.

Mentions: Crystals started growing in the capillaries 2 to 3 weeks after sample loading on the ground. The crystals we obtained are shown in Figure 2. In microgravity, we obtained rodlike crystals of the wild-type L-PGDS (1.0 × 0.1 × 0.1 mm3, Figure 2a) and the C65A mutant (0.5 × 0.1 × 0.1 mm3, Figure 2b) along the capillaries. While, on the ground, thin, platelike crystals of the wild-type L-PGDS (0.4 × 0.1 × 0.05 mm3, Figure 2c) and the C65A mutant (0.5 × 0.3 × 0.05 mm3, Figure 2d) were grown in the capillaries. Crystals of wild-type mouse L-PGDS were obtained for the first time.


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

Inaka K, Takahashi S, Aritake K, Tsurumura T, Furubayashi N, Yan B, Hirota E, Sano S, Sato M, Kobayashi T, Yoshimura Y, Tanaka H, Urade Y - Cryst Growth Des (2011)

(a) Crystals of wild-type L-PGDS grown in microgravity. (b) Crystals of the C65A mutant grown in microgravity. (c) Crystals of wild-type L-PGDS grown on the ground. (d) Crystals of the C65A mutant grown on the ground. The scale bars correspond to 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105485&req=5

fig2: (a) Crystals of wild-type L-PGDS grown in microgravity. (b) Crystals of the C65A mutant grown in microgravity. (c) Crystals of wild-type L-PGDS grown on the ground. (d) Crystals of the C65A mutant grown on the ground. The scale bars correspond to 100 μm.
Mentions: Crystals started growing in the capillaries 2 to 3 weeks after sample loading on the ground. The crystals we obtained are shown in Figure 2. In microgravity, we obtained rodlike crystals of the wild-type L-PGDS (1.0 × 0.1 × 0.1 mm3, Figure 2a) and the C65A mutant (0.5 × 0.1 × 0.1 mm3, Figure 2b) along the capillaries. While, on the ground, thin, platelike crystals of the wild-type L-PGDS (0.4 × 0.1 × 0.05 mm3, Figure 2c) and the C65A mutant (0.5 × 0.3 × 0.05 mm3, Figure 2d) were grown in the capillaries. Crystals of wild-type mouse L-PGDS were obtained for the first time.

Bottom Line: 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.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.The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0).

View Article: PubMed Central - PubMed

Affiliation: Maruwa Foods and Biosciences Inc. , 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus