Bayburt Timothy H, Sligar Stephen G
Beckman Institute for Advanced Science and Technology, Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6725-30. doi: 10.1073/pnas.062565599. Epub 2002 May 7.
The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.
膜蛋白在磷脂双分子层天然环境中的结构对于理解其生理功能至关重要，但通过实验实现却颇具难度。在本通讯中，我们描述了将一种膜锚定蛋白整合到支撑的磷脂双分子层中的过程。从肝脏内质网中溶解并纯化得到的细胞色素P450 2B4被整合到磷脂双分子层纳米结构中，并定向于表面以便通过原子力显微镜进行可视化观察。观察到单个P450分子从双分子层表面突出。通过分析力依赖的高度测量来定量蛋白质在双分子层表面上方的高度，避免了原子力显微镜探针使蛋白质变形的相关问题。作为探针 - 样品间距函数的原子力显微镜悬臂偏转测量结果表明，与N端膜锚定区域相对的P450顶部位于磷脂 - 水界面上方3.5纳米处。本文展示并讨论了该酶的取向模型，涉及膜相互作用以及与细胞色素P450还原酶的相互作用。
