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In Escherichia coli and other Gram-negative bacteria energy coupled outer membrane transporters 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, which is transduced by the ExbB-ExbD- TonB protein complex from the cytoplasmic membrane. Little is known about the structure and stoichiometry of this complex, which is required to elucidate the mechanisms of energy harvesting at the cytoplasmic membrane and concomitant energy transfer to the outer membrane transporters. We found that C-terminally His6 tagged ExbB and and Strep Tagged ExbD are as functional as wild type. We solubilized an ExbB oligomer and an ExbB-ExbD subcomplex from the cytoplasmic membrane with the help of the detergents decyl and undecyl maltoside. We have purified tagged ExbB oligomer and ExbB-ExbD complex by affinity chromatograph followed by size exclusion chromatography. We have characterized the protein complex in solution by Blue Native PAGE, size exclusion chromatography and small angle X-ray scattering (SAXS). All the methods indicated that there are 4-6 ExbB monomers in the complex. To understand the definite stoichiometry of the complexes we used laser- induced liquid bead ion desorption mass spectrometry (LILBID-MS). At moderate desorption laser energies we determined the oligomeric structure of ExbB to be mainly hexameric (ExbB6 ), with minor amounts of trimers (ExbB 3 ), dimers (ExbB 2 ), and monomers (ExbB 1 ). Under the same conditions ExbB-ExbD formed a complex consisting of ExbB6 ExbD1 , with a minor amount of ExbB 5 ExbD1 . At higher desorption laser intensities, ExbB 1 and ExbD1 and traces of ExbB 3 ExbD1, ExbB 2 ExbD1 , ExbB 1ExbD1, ExbB 3, and ExbB 2 were observed. Since the ExbB6 complex and the ExbB 6ExbD1 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 conclud that ExbB6 and ExbB 6 ExbD1 are subcomplexes on which the final complex including TonB is assembled.
