title: Purification, crystallization and preliminary X-ray diffraction studies of the ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.
abstract: ATP-binding cassette (ABC) transporters, also known as traffic ATPases, form a large family of integral membrane proteins responsible for the translocation of a variety of chemically diverse substrates across the lipid bilayers of cellular membranes of both prokaryotes and eukaryotes by the hydrolysis of ATP. The ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus, a homodimeric enzyme, was overexpressed in Escherichia coli and purified. Crystals were obtained using the microbatch-under-oil method at 291 K. X-ray diffraction data to 1.6 Å resolution were collected on SPring-8 beamline BL26B1. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a=54.94, b=78.63, c=112.96 Å. Assuming the presence of a dimer in the asymmetric unit gave a crystal volume per protein weight (VM) of 2.32 Å3 Da(-1) and a solvent content of 47%; this was consistent with the results of a dynamic light-scattering experiment, which showed a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of HisP from the Salmonella typhimurium ATP-binding subunit of an ABC transporter as a search model did not provide a satisfactory solution, indicating that the two ATP-binding subunits of ABC transporters have substantially different structures.
authors.affiliation: Department of Studies in Physics, University of Mysore, Mysore 570 006, India.
authors.forename: Mallappa
authors.initials: M
authors.lastname: Manjula
authors.forename: Kudigana J
authors.initials: KJ
authors.lastname: Pampa
authors.forename: Shankar
authors.initials: S
authors.lastname: Madan Kumar
authors.forename: Naoki
authors.initials: N
authors.lastname: Kunishima
authors.forename: Neratur K
authors.initials: NK
authors.lastname: Lokanath
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D056506:Geobacillus; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D021122:Protein Subunits