Lu Wei, Zhong Meng, Wei Yinan
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
Protein Pept Lett. 2011 Sep;18(9):863-71. doi: 10.2174/092986611796011446.
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.
AcrB是大肠杆菌中的一种内膜蛋白，是三聚体AcrA-AcrB-TolC（AcrAB-TolC）多药外排泵的一个组成部分。AcrAB-TolC复合物及其同源物在革兰氏阴性菌中高度保守，并且是许多病原体中赋予多药耐药性（MDR）的主要因素。在本研究中，我们开发了一种二硫键捕获方法，该方法可能揭示细胞膜天然条件下AcrB的构象变化。具体而言，我们在AcrB的周质结构域中创建了七对二硫键，可作为确定局部构象变化的探针。我们已经开发出一种严格的方案来测量天然条件下膜蛋白中二硫键形成的程度。通过检查不同距离分隔的半胱氨酸对中二硫键形成的程度，验证了该方法的严谨性。阻断-还原-标记方案与荧光标记相结合，使当前方法既方便、可靠又具有定量性。
