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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

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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 TonB147–239 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 P212121 (unit‐cell parameters a = 74.3, b = 82.4, c = 122.6 Å). The asymmetric unit consists of a single BtuB–TonB complex. Data sets have been collected to 2.1 Å resolution at a synchrotron beamline (APS SER‐CAT 22‐ID).
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Keywords: BtuB; E. coli; Gram‐negative bacteria; TonB; TonB‐dependent transporters; cyanocobalamin; membrane proteins; outer membrane transporters

Document Type: Research Article

Publication date: July 1, 2006

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