Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: Silane-polyethyleneglycol-lipid as a cushion and covalent linker
Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: Silane-polyethyleneglycol-lipid as a cushion and covalent linker
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DOI：
10.1016/s0006-3495(00)76392-2
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发表时间：
2000-09-01
影响因子：
3.4
通讯作者：
Tamm, LK
中科院分区：
文献类型：
--
作者：
Wagner, ML;Tamm, LK
支撑膜作为生物膜的模型以及作为研究膜蛋白和受体的结构和功能的生理基质越来越受到人们的关注。直接支撑在亲水性基底上的蛋白质-脂质双层的一个常见问题是完整膜蛋白与固体支撑物的非生理相互作用，其程度为它们不会在膜平面中扩散。为了缓解这些问题，我们已经开发了一种新的拴系聚合物支持的平面脂质双层系统，这使我们能够重建整体膜蛋白的横向移动的形式。我们支持脂质双层上的一个新设计的聚乙二醇垫，它提供了一个软的支持，并增加稳定性，共价键的膜支持石英或玻璃基板。随后通过全内反射和落射荧光显微镜观察双层的形成和形态，并通过光漂白后的荧光恢复监测双层中脂质和蛋白质的侧向扩散。当聚合物浓度保持略低于蘑菇-刷状转变时，观察到具有高横向脂质扩散系数（0.8-1.2 × 10（-8）cm（2）/s）的均匀双层。细胞色素B（5）和膜联蛋白V用作该系统中的第一个测试蛋白。当在直接负载在石英上的支撑双层中重构时，两种蛋白质在很大程度上是不移动的，其中移动的部分<25%。然而，在聚合物支撑的双层中观察到两个群体的横向移动的蛋白质。大约25%的细胞色素B（5）以近似1 x 10（-8）cm（2）/s的扩散系数扩散，50-60%以近似2 x 10（-10）cm（2）/s的扩散系数扩散。类似地，三分之一的膜联蛋白V扩散的扩散系数近似于3 × 10（-9）cm（2）/s，三分之二的膜联蛋白V扩散的扩散系数近似于4 × 10（-10）cm（2）/s。这些蛋白质与底层聚合物的相互作用的模型进行了讨论。
There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of similar to 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of similar to 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of similar to 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of similar to 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.