UMR 6022 CNRS, Université de Technologie de Compiègne, Compiègne, France.
PLoS One. 2011 Apr 20;6(4):e19101. doi: 10.1371/journal.pone.0019101.
Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a "trans" side, one to a few nanometer thick, located between the supporting surface and the membrane; and a "cis" side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a "cis" side corresponding to the extracellular milieu and a "trans" side marked by a key cytosolic signaling protein, calmodulin.
METHODOLOGY/PRINCIPAL FINDINGS: We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side).
The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules.
固载仿生膜模型对于膜蛋白生物化学和生物技术是非常重要的工具。迄今为止所描述的支撑膜系统由脂质双层组成，这些双层要么与一个表面连接，要么不连接，这个表面将两个隔室分隔开：一个“跨膜”侧，厚度为一到几个纳米，位于支撑表面和膜之间；另一个“胞质”侧，位于合成膜之上，暴露于大量介质中。我们在这里描述了一种新的仿生设计，它由一个固载双层膜组成，该双层膜组装在一个带有特定细胞内蛋白质标记的表面上。这种多层仿生组装在两个不同隔室之间形成了连续但可流动的磷脂屏障，具有真正生物膜的基本特征：一个“胞质”侧对应细胞外环境，另一个“跨膜”侧则由一种关键的胞质信号蛋白——钙调蛋白标记。
方法/主要发现：我们建立并验证了实验条件，以构建一种多层结构，该结构由一个组装在钙调蛋白衍生表面上的平面固载双层组成。我们证明了以下几点：（i）在跨膜侧接枝的钙调蛋白分子完全具有结合和激活钙调蛋白依赖性酶——百日咳博德特氏菌的腺苷酸环化酶的功能；（ii）组装的双层形成了一个连续的、蛋白质不可渗透的边界，将下面的钙调蛋白（跨膜侧）与上面的介质（胞质侧）完全分隔开。
这里描述的固载双层结构的简单性和稳健性应该有助于构建包含膜嵌入蛋白和关键细胞质成分的仿生膜模型。这种仿生结构也将成为研究蛋白质或其他大分子跨生物膜转运的有吸引力的工具。
