Department of Chemistry, Washington State University, PO Box 644630, Pullman, Washington 99164-4630, United States.
Langmuir. 2011 Jan 4;27(1):320-7. doi: 10.1021/la104133m. Epub 2010 Dec 8.
A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a “single-cushion” POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains (<r(C)2> 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC−PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.
与含有蛋白质的支撑双层组装相关的一个主要问题是蛋白质在掺入后是否仍然保持活性。主要关注的问题是，与固体载体的强烈相互作用会使蛋白质失活。为了解决这个问题，已经将一种大型跨膜蛋白，5-羟色胺受体 5HT(3A)，掺入到几种越来越复杂的支撑双层膜组装体中。5HT(3A)受体在膜的两侧都有大的细胞外结构域，这可能会导致强烈的相互作用。双层组装体包括一个简单的 POPC（1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱）支撑的平面双层、一个在膜和支撑物之间有 PEG（聚乙二醇）层的“单缓冲垫”POPC 双层、以及一个具有 PEG 层和 BSA（牛血清白蛋白）层的“双缓冲垫”POPC 双层。单缓冲垫系统旨在将双层从表面抬起，而双缓冲垫系统旨在同时抬起膜并钝化固体支撑物。与之前报道的工作一样，通过荧光漂白后荧光恢复的整体测量（FRAP）测量的蛋白质迁移率非常低，尤其是在双缓冲垫系统中。但是，荧光 5HT(3A)分子的单粒子跟踪表明，双缓冲垫系统中的单个蛋白质具有相当高的局部迁移率，但在小的围栏域（<r(C)> 450nm）内受到空间限制。与简单的 POPC 膜和单缓冲垫 POPC−PEG 膜的比较表明，BSA 既能最小化与固体支撑物的相互作用，又能形成围栏，降低大跨膜蛋白的长程（整体平均）迁移率。这些结果表明，在双缓冲垫组装体中，具有大的细胞外结构域的蛋白质尽管具有低的体扩散常数，但可能仍然保持活性且未受干扰。
