PMID:21905676
| Citation |
Pramanik, A, Hauf, W, Hoffmann, J, Cernescu, M, Brutschy, B and Braun, V (2011) Oligomeric structure of ExbB and ExbB-ExbD isolated from Escherichia coli as revealed by LILBID mass spectrometry. Biochemistry 50:8950-6 |
|---|---|
| Abstract |
Energy-coupled transporters in the outer membrane of Escherichia coli and other Gram-negative bacteria allow the entry of scarce substrates, toxic proteins, and bacterial viruses (phages) into the cells. The required energy is derived from the proton-motive force of the cytoplasmic membrane, which is coupled to the outer membrane via the ExbB-ExbD-TonB protein complex. Knowledge of the structure of this complex is required to elucidate the mechanisms of energy harvesting in the cytoplasmic membrane and energy transfer to the outer membrane transporters. Here we solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the detergent undecyl maltoside. Using laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), we determined at moderate desorption laser energies the oligomeric structure of ExbB to be mainly hexameric (ExbB(6)), with minor amounts of trimeric (ExbB(3)), dimeric (ExbB(2)), and monomeric (ExbB(1)) oligomers. Under the same conditions ExbB-ExbD formed a subcomplex consisting of ExbB(6)ExbD(1), with a minor amount of ExbB(5)ExbD(1). At higher desorption laser intensities, ExbB(1) and ExbD(1) and traces of ExbB(3)ExbD(1), ExbB(2)ExbD(1), ExbB(1)ExbD(1), ExbB(3), and ExbB(2) were observed. Since the ExbB(6) complex and the ExbB(6)ExbD(1) complex remained stable during solubilization and subsequent chromatographic purification on nickel-nitrilotriacetate agarose, Strep-Tactin, and Superdex 200, and during native blue gel electrophoresis, we concluded that ExbB(6) and ExbB(6)ExbD(1) are subcomplexes on which the final complex including TonB is assembled. |
| Links |
PubMed Online version:10.1021/bi2008195 |
| Keywords |
Biochemistry/methods; Chromatography/methods; Cloning, Molecular; Dimerization; Electrophoresis, Polyacrylamide Gel/methods; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Magnetic Resonance Spectroscopy/methods; Maltose/chemistry; Mass Spectrometry/methods; Models, Molecular; Molecular Conformation; Plasmids/metabolism |
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