TY - JOUR
T1 - Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli
T2 - A Biochemical and Structural Case Study
AU - Sverzhinsky, Aleksandr
AU - Qian, Shuo
AU - Yang, Lin
AU - Allaire, Marc
AU - Moraes, Isabel
AU - Ma, Dewang
AU - Chung, Jacqueline W.
AU - Zoonens, Manuela
AU - Popot, Jean Luc
AU - Coulton, James W.
N1 - Publisher Copyright:
© 2014, Springer Science+Business Media New York.
PY - 2014/10/14
Y1 - 2014/10/14
N2 - 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.
AB - 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.
KW - Amphipol
KW - Detergent
KW - EM
KW - Membrane protein complex
KW - SAXS/SANS
UR - http://www.scopus.com/inward/record.url?scp=84911003599&partnerID=8YFLogxK
U2 - 10.1007/s00232-014-9678-4
DO - 10.1007/s00232-014-9678-4
M3 - Article
C2 - 24862870
AN - SCOPUS:84911003599
SN - 0022-2631
VL - 247
SP - 1005
EP - 1018
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
IS - 9-10
ER -