Crystallization and preliminary X-ray studies of ferric uptake regulator fromMagnetospirillum gryphiswaldense

Abstract

Magnetosomes in magnetotactic bacteria have been widely used in studies of magnetic domains and in commercial applications. The iron content of magnetotactic bacteria is $100 times higher than that of Escherichia coli. Magnetospirillum gryphiswaldense MSR-1 can still take up iron even at high intracellular concentrations. Ferric uptake regulator (Fur) is a global ironresponsive regulator that affects magnetosome formation, iron transport and oxygen metabolism. However, the mechanism of iron uptake and homeostasis by M. gryphiswaldense MSR-1 Fur is not clear. Here, the expression, purification and crystallization of apo and SeMet Fur from M. gryphiswaldense MSR-1 are reported. The crystals belonged to space group C2. Matthews coefficient analysis and size-exclusion chromatography showed that the asymmetric unit probably contains one dimer of Fur. Diffraction data were optimized to 1.58 Å resolution for apo Fur and to 1.9 Å resolution for SeMet Fur.

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

“…Proteins were expressed in E. coli strain BL21 (DE3). Full-length and selenomethionine-containing Fur proteins were overexpressed and purified according to the reference 44 . Mutant proteins were overexpressed and purified by the same procedure.…”

“…Crystals of apo-Fur and selenomethionyl-derivative proteins were obtained using a reservoir consisting of 1.8 M (NH 4 ) 2 SO 4 and 0.1 M sodium citrate at pH 5.5, 1.4 M (NH 4 ) 2 SO 4 and 0.1 M sodium citrate at pH 5.5, respectively 44 . To obtain holo-Fur for crystallization, MnCl 2 (final concentration: 1 mM) was added to the buffer, and the protein was concentrated to 10 mg ml −1 .…”