In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin.
The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.
Bradburne
J A
JA
School of Biology, Georgia Institute of Technology, Atlanta 30332.
Godfrey
P
P
Choi
J H
JH
Mathis
J N
JN
D000614:Aminocaproates; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D004952:Esters; D012680:Sensitivity and Specificity; D013388:Succinimides
D000614:Aminocaproates; D001425:Bacterial Outer Membrane Proteins; D004952:Esters; D013388:Succinimides; C061443:sulfosuccinimidyl 6-(biotinamido)hexanoate; D001710:Biotin; C048592:biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester