Purification and structural and functional characterization of FhuA, a transporter of the Escherichia coli outer membrane.
The Escherichia coli outer membrane ferrichrome transporter FhuA was purified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside). The amount of dodecyl maltoside bound to the protein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodecyl maltoside complex (Rs = 4.2 nm) were determined using size exclusion chromatography. Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the protein was monomeric. The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for the complex. Sedimentation experiments gave values for the molecular mass of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes radius in complete agreement with those calculated from size exclusion chromatography. The circular dichroism spectrum indicated a 51% beta-sheet content. Functionality of the purified protein was assessed from fluorescence measurements using the DNA probe YO-PRO-1. Interaction of nM concentrations of FhuA with bacteriophage T5 resulted in the release of 90 +/- 8% of the phage DNA. The limiting step in DNA ejection was binding of the phage to its receptor. Release of DNA took place in a few seconds. Ferrichrome (0.8 microM) competed with the phage for binding to FhuA and prevented DNA ejection.
Laboratoire des Biomembranes, URA CNRS 1116, Université Paris-Sud, Orsay, France.
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Boulanger
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le Maire
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Bonhivers
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Dubois
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Desmadril
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Letellier
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D001667:Binding, Competitive; D001692:Biological Transport; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003090:Coliphages / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011991:Receptors, Virus / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure*; D014461:Ultracentrifugation
D001425:Bacterial Outer Membrane Proteins; D004279:DNA, Viral; D003902:Detergents; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D046911:Macromolecular Substances; D011991:Receptors, Virus; D005291:Ferrichrome