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

Abstract

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to PGD2 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.

Effect of an External Electric Field on the Kinetics of Dislocation-Free Growth of Tetragonal Hen Egg White Lysozyme Crystals

“…The growth of high-quality single crystals of proteins has been intensively pursued using magnetic fields [3][4][5][6][7][8][9], microgravity [10][11][12][13][14][15][16], solution flow [17][18][19][20], and gel as a growth host media [21][22][23][24][25][26][27][28]. A growth technique mediated by screw dislocations under low supersaturation has also been proposed [29][30][31][32].…”

Technique for High-Quality Protein Crystal Growth by Control of Subgrain Formation under an External Electric Field

“…However, the growth of high-quality single crystals of proteins is difficult, which is an impediment to accurate elucidation of the 3D structures of protein molecules. Therefore, the establishment of crystallization techniques to obtain high-quality single crystals of proteins has been intensively pursued using magnetic fields [1][2][3][4][5][6][7][8], microgravity [9][10][11][12][13][14][15][16], electric fields [17][18][19][20][21], solution flow [22][23][24][25] and gel as a growth host media [26][27][28][29][30][31][32]. In this article, the improvement of the crystal quality of tetragonal hen egg white (HEW) lysozyme crystals as a model protein is demonstrated by applying an external electric field.…”

Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry

“…[1][2][3][4][5][6][7][8] The experiments are of interest because a significant number of space-grown protein crystals were found to exhibit better resolution in X-ray diffraction studies as compared to the crystals grown on Earth. 9 However, the origin of such improved resolution in space environment has remained elusive, although there are many speculative discussions.…”