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 chan nel that is closed by a loop, tentatively designated the 'gating loop' , which is also the principal binding site for all FhuA ligands. In th is report, it is shown by in vivo labeling that the two cysteines in t he gating loop form a disulfide bridge, and they react weakly after re duction with biotin-maleimide, as determined by streptavidin-beta-gala ctosidase bound to biotin. The two cysteines close to the C-terminus o f FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS. Replacement of the existi ng cysteines by serine did not alter the sensitivity of cells to the F huA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supp orted 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 essenti al for maintaining the conformation of FhuA. The C-terminal cysteines are in the interior of FhuA and are also not important for the structu re of FhuA. The method used allows the identification of free cysteine s and disulfides in surface exposed protein regions.