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The role of CAPS buffer in expanding the crystallization space of the nucleotide-binding domain of the ABC transporter haemolysin B from Escherichia coli

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Abstract:

Nucleotide-binding domains (NBDs), which are roughly 27 kDa in size, are conserved components of the large family of ABC (ATP-binding cassette) transporters, which includes importers and exporters. NBDs, or ABC-ATPases, supply energy for the translocation of a vast range of substrates across biological membranes. Despite their hydrophilic sequence, many NBDs readily associate in some way with membranes but demonstrate extreme instability in solution upon separation from the complete transporter. Conditions that stabilized the purified ABC domain of the Escherichia coli haemolysin A (HlyA) transporter were developed. This allowed the screening of unlimited crystallization conditions in the presence of different substrates, the performance of reproducible functional assays and the protection of 50 mg ml−1 protein from precipitation on ice for months. As a result, it became possible to obtain crystals of HlyB-NBD in the presence of ADP and ATP that were suitable for X-ray analysis. Although the focus of these investigations was placed on HlyB-NBD, the strategy described here can be directly transferred to other proteins that display instability in solution.

Keywords: ABC transporters; CAPS buffer; haemolysin B; nucleotide-binding domains

Document Type: Research Article

DOI: http://dx.doi.org/10.1107/S0907444904007437

Publication date: June 1, 2004

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