An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.
An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.
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DOI：
10.1194/jlr.d002493
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发表时间：
2010-05
影响因子：
6.5
通讯作者：
Ren G
中科院分区：
文献类型：
--
作者：
Zhang L;Song J;Newhouse Y;Zhang S;Weisgraber KH;Ren G
载脂蛋白E(ApoE)是外周和中枢神经系统脂蛋白的主要蛋白质组分之一，通过与低密度脂蛋白受体家族成员的相互作用来调节胆固醇代谢。了解载脂蛋白E功能的关键之一是确定载脂蛋白E脂结合形式的结构。负染(NS)电子显微镜(EM)是研究载脂蛋白E脂结合形式的结构和形态的一种简单而快速的方法。然而，使用传统NS方案的一个伪影是apoE·磷脂颗粒形成规则。在这项研究中，我们使用冷冻电子显微镜(Cryo-EM)观察了冷冻水合天然状态下的apoE4·棕榈酰油酰磷脂酰磷脂酰胆碱(POPC)颗粒。通过比较不同NS方案产生的颗粒大小和形状与Cryo-EM产生的颗粒尺寸和形状，我们提出了一种检测apoE4·POPC颗粒的优化方案。统计分析表明，优化后的方案与冷冻-EM法相比，颗粒大小相差不到5%，颗粒形状相似。使用这种优化的方案产生的高对比度和精细的粒子图像有助于载脂蛋白E脂结合形式的结构研究。
Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE•phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4•palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4•POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.