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.

SUBMITTER: Manjula M 

PROVIDER: S-EPMC3515392 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature