Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop.
The multifunctional FhuA protein of Escherichia coli K-12 forms a channel that is closed by a loop, tentatively designated the 'gating loop', which is also the principal binding site for all FhuA ligands. In this report, it is shown by in vivo labeling that the two cysteines in the gating loop form a disulfide bridge, and they react weakly after reduction with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. The two cysteines close to the C-terminus of FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS. Replacement of the existing cysteines by serine did not alter the sensitivity of cells to the FhuA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supported growth on ferrichrome as sole iron source. The cysteines in the gating loop play no specific functional role; they are largely buried in the interior of the loop, and the disulfide bridges are not essential for maintaining the conformation of FhuA. The C-terminal cysteines are in the interior of FhuA and are also not important for the structure of FhuA. The method used allows the identification of free cysteines and disulfides in surface exposed protein regions.
Bös
C
C
Universität Tübingen, Germany.
Braun
V
V
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001710:Biotin; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005291:Ferrichrome; D006358:Hot Temperature; D008024:Ligands; D008301:Maleimides; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics; D013439:Sulfhydryl Reagents
D001425:Bacterial Outer Membrane Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008024:Ligands; D008301:Maleimides; D011991:Receptors, Virus; D013439:Sulfhydryl Reagents; D005291:Ferrichrome; C043592:maleimide; D001710:Biotin; D003545:Cysteine