Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks
Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks
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DOI：
10.1073/pnas.062565599
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发表时间：
2002-05-14
影响因子：
11.1
通讯作者：
Sligar, SG
中科院分区：
文献类型：
--
作者：
Bayburt, TH;Sligar, SG
膜蛋白在磷脂双层的天然环境中的结构对于理解生理功能至关重要，但很难通过实验实现。在这篇文章中，我们描述了将膜锚定蛋白掺入到支持的磷脂双层中。将从肝内质网溶解和纯化的细胞色素P450 2B 4掺入磷脂双层纳米结构中，并通过原子力显微镜在表面上取向以进行可视化。观察到单个P450分子从双层表面突出。与变形的蛋白质的原子力显微镜探针的问题，避免了通过分析力依赖的高度测量，以定量的蛋白质的高度以上的双层表面。作为探针-样品分离的函数的原子力显微镜悬臂梁偏转的测量揭示，P450的N-末端膜锚区域对面的顶部位于磷脂-水边界上方3.5纳米。模型的酶的取向提出和讨论有关膜的相互作用和细胞色素P450还原酶的相互作用。
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.