Crystallization and preliminary X-ray crystallographic analysis of the Escherichia coli outer membrane cobalamin transporter BtuB in complex with the carboxy-terminal domain of TonB.
The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147-239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB(147-239) was formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B12) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C8E4 (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2(1)2(1)2(1) (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 angstroms). The asymmetric unit consists of a single BtuB-TonB complex. Data sets have been collected to 2.1 angstroms resolution at a synchrotron beamline (APS SER-CAT 22-ID).
Shultis
David D
DD
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.
Purdy
Michael D
MD
Banchs
Christian N
CN
Wiener
Michael C
MC
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C489181:tonB protein, E coli; D014805:Vitamin B 12; D002118:Calcium