title: A reporter platform for the monitoring of in vivo conformational changes in AcrB.
abstract: AcrB is an inner membrane protein in Escherichia coli that is a component of a triplex AcrA-AcrB-TolC (AcrAB-TolC) multidrug efflux pump. The AcrAB-TolC complex and its homologues are highly conserved among Gram-negative bacteria and are major players in conferring multidrug resistance (MDR) in many pathogens. In this study we developed a disulfide trapping method that may reveal AcrB conformational changes under the native condition in the cell membrane. Specifically, we created seven disulfide bond pairs in the periplasmic domain of AcrB, which can be used as probes to determine local conformational changes. We have developed a rigorous protocol to measure the extent of disulfide bond formation in membrane proteins under the native condition. The rigorousness of the method was verified through examining the extent of disulfide bond formation in Cys pairs separated by different distances. The blocking-reducing-labeling scheme combined with fluorescence labeling made the current method convenient, reliable, and quantitative.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000737:chemistry*; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis*; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D003545:Cysteine; D004220:Disulfides; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli