TY - JOUR
T1 - A reporter platform for the Monitoring of in Vivo conformational changes in AcrB
AU - Lu, Wei
AU - Zhong, Meng
AU - Wei, Yinan
PY - 2011/9
Y1 - 2011/9
N2 - 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.
AB - 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.
KW - Disulfide trapping
KW - Protein tertiary and quaternary structures
UR - http://www.scopus.com/inward/record.url?scp=79959419297&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79959419297&partnerID=8YFLogxK
U2 - 10.2174/092986611796011446
DO - 10.2174/092986611796011446
M3 - Article
C2 - 21529338
AN - SCOPUS:79959419297
SN - 0929-8665
VL - 18
SP - 863
EP - 871
JO - Protein and Peptide Letters
JF - Protein and Peptide Letters
IS - 9
ER -