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 p urified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside). The amount of dodecyl maltoside bound to the prot ein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodec yl maltoside complex (R(S) = 4.2 nm) were determined using size exclus ion chromatography, Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the pro tein was monomeric. The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for th e complex, Sedimentation experiments gave values for the molecular mas s of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes r adius in complete agreement with those calculated from size exclusion chromatography, The circular dichroism spectrum indicated a 51% beta-s heet 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 relea se of 90 +/- 8% of the phage DNA, The limiting step in DNA ejection wa s binding of the phage to its receptor. Release of DNA took place in a few seconds. Ferrichrome (0.8 mu M) competed with the phage for bindi ng to FhuA and prevented DNA ejection.