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A Reporter Platform for the Monitoring of In Vivo Conformational Changes in AcrB

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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.

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Keywords: Disulfide trapping; Methicillin-resistant Staphylococcus aureus (MRSA); circular dichroism (CD); conformational changes; efflux pump; efflux pumps; fluorescence intensity; fluorescent labeling; gel electrophoresis; minimum inhibitory concentrations; multidrug resistance (MDR); mutagenesis; periplasmic domain; plasmids; prokaryotic cells; protein tertiary; quaternary structures; radioactive probes

Document Type: Research Article

Publication date: September 1, 2011

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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