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 Gramnegative 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 blockingreducing-labeling scheme combined with fluorescence labeling made the current method convenient, reliable, and quantitative.
| Original language | English |
|---|---|
| Pages (from-to) | 863-871 |
| Number of pages | 9 |
| Journal | Protein and Peptide Letters |
| Volume | 18 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 2011 |
Keywords
- Disulfide trapping
- Protein tertiary and quaternary structures
ASJC Scopus subject areas
- Structural Biology
- Biochemistry