Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.
We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.
The Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Chuck R
CR
Smallwood
Amparo Gala
AG
Marco
Qiaobin
Q
Xiao
Vy
V
Trinh
Salete M C
SM
Newton
Phillip E
PE
Klebba
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005452:Fluoresceins; D005453:Fluorescence; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; C489181:tonB protein, E coli; C037041:fluorescein 5-maleimide