Kreimer D I, Chai K P, Ferro-Luzzi Ames G
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720, USA.
Biochemistry. 2000 Nov 21;39(46):14183-95. doi: 10.1021/bi001066+.
The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.
鼠伤寒沙门氏菌组氨酸通透酶（一种ABC转运蛋白，即运输ATP酶）的膜结合复合物由两种膜蛋白HisQ和HisM以及两个相同的ATP水解蛋白HisP拷贝组成。我们开发了一种技术，可定量监测完整复合物内的巯基修饰水平，并利用该技术研究HisP亚基在复合物内的行为是否相同。我们在此表明，它们与各种硫醇特异性试剂的相互作用不同，这表明尽管它们相同，但排列不对称。讨论了这种不对称的可能基础。我们还使用硫醇特异性试剂、荧光测量和圆二色光谱分析了活性循环各个阶段构象变化的发生情况。靠近ATP结合口袋的Cys-51反映了ATP结合时的构象变化，但不参与信号传导和转运所涉及的变化。后者会导致二级结构改变，如α螺旋的变化所示；三级结构也会发生改变，如荧光研究所示。
