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J Membr Biol. 2014 Oct;247(9-10):1005-18. doi: 10.1007/s00232-014-9678-4. Epub 2014 May 27.

Amphipol-trapped ExbB-ExbD membrane protein complex from Escherichia coli: a biochemical and structural case study.

Author information

1
Department of Microbiology and Immunology, McGill University, Montreal, H3A 2B4, QC, Canada.

Abstract

Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4-ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.

PMID:
24862870
DOI:
10.1007/s00232-014-9678-4
[Indexed for MEDLINE]

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