• analytical ultracentrifugation;
  • β barrel;
  • ferrichrome transport;
  • membrane protein;
  • signal transduction


  1. Top of page
  2. Abstract
  3. References

The channel-forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution. The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis. Analytical ultracentrifugation revealed a monodisperse species (S20,w= 3.8 S) with a mass of 77 800 ± 3200 Da. The properties of ligand binding, determined by two independent methods, revealed one binding site/ monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5. This strongly suggests that oligomeric species are present. Cross-linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species. The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ.


decyl maltoside




n-octyl β-d-glucopyranoside


periodic acid-Schiff staining


  1. Top of page
  2. Abstract
  3. References
  • 1
    Neilands, J. B. (1981) Microbial iron compounds, Annu. Rev. Biochem. 50, 115131.
  • 2
    Nau, C. D. & Konisky, J. (1989) Evolutionary relationship between TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene, J. Bacteriol. 171, 10411047.
  • 3
    Guerinot, M. L. (1994) Microbial iron transport, Annu. Rev. Microhiol. 48, 743112.
  • 4
    Kadner, R. J. (1990) Vitamin B12 transport in Escherichia coli:energy coupling between membranes, Mol. Microbiol. 4, 20272033.
  • 5
    Braun, V. (1995) Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins, FEMS Microbiol. Rev. 16, 295301.
  • 6
    Klebba, P. E., Rutz, J. M., Liu, J. & Murphy, C. K. (1993) Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope, J. Bioenerg. Biornernbl: 25, 603611.
  • 7
    Postle, K. (1990) TonB and the Gram-negative dilemma, Mol. Microbiol. 4, 20192025.
  • 8
    Braun, V., Hancock, R. E. W., Hantke, K. & Hartmann, A. (1976) Functional organization of the outer membrane of Escherichia coli: phage and colicin receptors as components of iron uptake systems, J. Supramol. Struct. 5, 3758.
  • 9
    Coulton, J. W., Mason, P. & DuBow, M. S. (1983) Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12, J. Bacteriol. 156, 13151321.
  • 10
    Fecker, L. & Braun, V. (1983) Cloning and expression of the flugenes involved in iron(III)-hydroxamate uptake by Escherichia coli, J. Bacteriol. 156, 13012314.
  • 11
    Menichi, B. & Buu, A. (1983) Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli, J. Bacteriol. 154, 130138.
  • 12
    Koebnik, R. & Braun, V. (1993) Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the Fhua outer membrane receptor of Escherfchia coli K-12, J. Bacteriol. 175, 826839.
  • 13
    Cowan, S. W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R. A., Jansonius, J. N. & Rosenbusch, J. P. (1992) Crystal structures explain functional properties of two E. coli porins, Nature 358, 127133.
  • 14
    Schirmer, T., Keller, T. A., Wang, Y. F. & Rosenbusch, J. P. (1995) Structural basis for sugar translocation through maltoporin channels at 3.1 Å resolution, Science 267, 512514.
  • 15
    Bonhivers, M., Ghazi, A., Boulanger, P. & Letellier, L. (1996) FhuA, a transporter of the Escherichiu coli outer membrane, is converted into a channel upon binding of bacteriophage T5, EMBO J. 15, 18501856.
  • 16
    Locher, K. P. & Rosenbusch, J. P. (1997) Modeling ligand-gated receptor activity: FhuA-mediated ferrichrome efflux from lipid vesicles triggered by Phage T5, J. Biol. Chem. 272, 14481451.
  • 17
    Monod, J., Wyman, J. & Changeux, J.-P. (1965) On the nature of allosteric transitions: a plausible model, J. Mol. Biol. 12, 88118.
  • 18
    Schlessinger, J. (1988) Signal transduction by allosteric receptor oligomerization, Trends Biochem. Sci. 13, 443447.
  • 19
    Lemmon, M. A. & Schlessinger, J. (1994) Regulation of signal transduction and signal diversity by receptor oligomerization, Trends Biochem. Sci. 19, 459463.
  • 20
    Landau, E. M. & Rosenbusch, J. P. (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins, Proc. Natl Acad. Sci. USA 93, 1453214535.
  • 21
    Hoffmann, H., Fischer, E., Kraut, H. & Braun, V. (1986) Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12, J. Bacteriol. 166, 404411.
  • 22
    Garavito, R. M. & Rosenbusch, J. P. (1986) Isolation and crystallization of bacterial porin, Methods Enzymol. 125, 309328.
  • 23
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4, Nature 227, 680685.
  • 24
    Boettcher, C. J. F., Van Gent, C. M. & Fries, C. (1961) A rapid and sensitive submicro phosphorous determination, Anal. Chim. Acta 24, 203204.
  • 25
    Skipski, V. P. & Barclay, M. (1969) Thin-layer chromatography of lipids, Methods Enzymol. 14, 530598.
  • 26
    Azem, A., Shaked, I., Rosenbusch, J. P. & Daniel, E. (1995) Crosslinking of porin with glutardialdehyde: a test for the adequacy of premises of cross-linking theory, Biochim. Biophys. Acta 1243, 151156.
  • 27
    Spies, M., Hauser, H., Rosenbusch, J. P. & Semenza, G. (1981) Hydrodynamic properties of phospholipid vesicles and of sucrase isomaltase-phospholipid vesicles, J. Biol. Chem. 256, 89778982.
  • 28
    Weaver, C. A. & Konisky, J. (1980) tonB-independent ferrichrome mediated iron transport in Escherichia coli spheroplasts, J. Bacteriol. 143, 15131518.
  • 29
    Rosenbusch, J. P. & Griffin, J. G. (1973) Carbamyl phosphate binding to aspartate transcarbamylase, J. Biol. Chem. 248, 50635066.
  • 30
    Prehm, P., Szrim, S., Jann, B. & Jann, K. (1975) Cell-wall lipopoly saccharide from Escherichia coli B, Eur. J. Biochem. 56, 4155.
  • 31
    Rosenbusch, J. P., Steven, A. C., Alkan, M. & Regenass, M. (1980) Matrix porin: a periodically arranged protein in the outer membrane of Escherichia coli, in: Electron microscopy at molecular dimensions (Baumeister, W. & Vogell, W., eds) pp. 110, Springer Verlag, Heidelberg-New York .
  • 32
    Garavito, R. M., Jenkins, J., Jansonius, J. N., Karlsson, R. & Rosenbusch, J. P. (1983) X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes, J. Mol. Biol. 164, 313327.
  • 33
    Buehler, L. (1989) Porins of Escherichia coli. Properties of the ion channels of porin B, porin F, porin C and phosphoporin, PhD Thesis, University of Basel, Basel, Switzerland.
  • 34
    Scatchard, G. (1949) The attractions of proteins for small molecules and ions, Ann. NY Acad. Sci. 51, 660672.
  • 35
    Hill, T. L. (1944) Relative free energies and dissociation constants of macroscopic ions, J. Phys. Chem. 48, 101111.
  • 36
    Rosenbusch, J. P. (1974) Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptido-glycan and unusual dodecyl sulfate binding, J. Biol. Chem. 249, 80198029.
  • 37
    Boulanger, P., Le Maire, M., Bonhivers, M., Dubois, S., Desmadril, M. & Letellier, L. (1996) Purification and structural and functional characterization of FhuA, a transporter of the Escherichia coli outer membrane, Biochemistry 35, 1421614224.
  • 38
    Nakamura, K. & Mizushima, S. (1976) Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12, J. Biochem. (Tokyo) 80, 14111422.
  • 39
    de Vos, A. M., Ultsch, M. & Kossiakoff, A. A. (1992) Human growth hormone and extracellular domain of its receptor: crystal structure of the complex, Science 255, 306312.
  • 40
    Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H. & Lesslauer, W. (1993) Crystal structure of the soluble human 55 kd TNF receptor-human TNF-beta complex: Implications for TNF receptor activation, Cell 73, 431445.
  • 41
    Cunningham, B. C., Ultsch, M., de Vos, A. M., Mulkerrin, M. G., Clauser, K. R. & Wells, J. A. (1991) Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule, Science 254, 821825.
  • 42
    Chapman, D. & Benga, G. (1984) Biomembrane fluidity-studies of model and natural biomembranes, in Biological membranes (Chapman, D., ed.) pp. 156, Academic Press Inc., London .