Zhu Yi, Guo Tiannan, Sze Siu Kwan
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Methods Mol Biol. 2011;790:287-303. doi: 10.1007/978-1-61779-319-6_22.
Although the snapshots of different in vitro conformational states have been intensively studied, current techniques such as nuclear magnetic resonance, X-ray crystallography, and electron microscope method cannot probe the in vivo conformational movements of integral membrane proteins on cell surfaces. Here, we describe a hydroxyl radical protein footprinting coupled to a mass spectrometry detection technique to probe the structural dynamics of a membrane protein directly on the native cell surface. This method uses in situ generation of hydroxyl radicals to oxidize and covalently modify integral membrane proteins on the cell surface. To explain this technique in detail, we use the porin OmpF as an example, although the method may be applied to study any membrane protein. Footprinting results show that the surface mapping data of OmpF are consistent with its current crystallographic structure. In addition, this technique also enables the detection of in vivo voltage gating of porin OmpF for the first time. This novel cell surface footprinting method coupled with MS analysis can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell.
尽管不同体外构象状态的快照已得到深入研究，但诸如核磁共振、X射线晶体学和电子显微镜方法等当前技术无法探测细胞表面整合膜蛋白的体内构象运动。在此，我们描述了一种与质谱检测技术相结合的羟基自由基蛋白足迹法，以直接在天然细胞表面探测膜蛋白的结构动力学。该方法利用原位产生的羟基自由基来氧化并共价修饰细胞表面的整合膜蛋白。为详细解释此技术，我们以孔蛋白OmpF为例，尽管该方法可应用于研究任何膜蛋白。足迹分析结果表明，OmpF的表面图谱数据与其当前的晶体结构一致。此外，该技术还首次实现了对孔蛋白OmpF体内电压门控的检测。这种结合质谱分析的新型细胞表面足迹法可能是研究活细胞膜蛋白结构动力学的一种潜在有效方法。
