TAILIEUCHUNG - Báo cáo Y học: Diffusion through channel derivatives of the Escherichia coli FhuA transport protein

FhuA is amultifunctional protein in the outer membrane of Escherichia colithat actively transports [Fe 3+ ]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, /80 and UC-1, and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infectionbyphageT5. | Eur. J. Biochem. 269 4948-4959 2002 FEBS 2002 doi Diffusion through channel derivatives of the Escherichia coli FhuA transport protein Michael Braun1 Helmut Killmann1 Elke Maier2 Roland Benz2 and Volkmar Braun1 1 MikrobiologieỊMembranphysiologie Universitat Tubingen Germany 2Lehrstuhl fur Biotechnologie Theodor-Boveri-Institut Biozentrum Universitat Wurzburg Germany FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports Fe3 ferrichrome the antibiotics albomycin and rifamycin CGP 4832 and mediates sensitivity of cells to the unrelated phages T5 T1 80 and UC-1 and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5. The FhuA crystal structure reveals 22 antiparallel transmembrane b-strands that form a b-barrel which is closed by a globular N-terminal domain. FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain residues 5-160 is excised and supports weakly unspecific diffusion of substrates across the outer membrane. Here it is shown that isolated FhuAA5-160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels. The double mutant FhuAA5-160 A322-336 lacking in addition to the globular domain most of the large surface loop 4 whiich pa 11al lly constricts ithe channel entrance displayed an increased single-channel conductance but formed no stable channels. It transported in vivo Fe3 ferrichrome with 45 of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics. In contrast a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335-355 comprising one-third of surface loop 4 and half of the transmembrane b-strand 8 formed stable channels in lipid .

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