Berquand Alexandre, Lévy Daniel, Gubellini Francesca, Le Grimellec Christian, Milhiet Pierre-Emmanuel
Institut Curie, UMR-CNRS 168 and LRC-CEA 34V, Paris Cedex 05, France.
Ultramicroscopy. 2007 Oct;107(10-11):928-33. doi: 10.1016/j.ultramic.2007.04.008. Epub 2007 Apr 27.
Reconstitution of transmembrane proteins by direct incorporation into supported lipid bilayers (SLBs) is a new method to provide suitable samples for high-resolution atomic force microscopy (AFM) analysis of membrane proteins. First experiments have reported successful incorporation of proteins into detergent-destabilized SLBs. Here, we analyzed by AFM the incorporation of membrane proteins in the presence of calcium, a divalent cation functionally important for several membrane proteins. Using lipid-phase-separated membranes, we first show that calcium strongly stabilizes the SLBs decreasing the insertion of low cmc detergents, dodecyl-beta-maltoside, dodecyl-beta-thiomaltoside, and N-hexadecylphosphocholine (Fos-Choline-16) and further insertion of proteins. However, high yield of protein insertion is recovered in the presence of calcium by increasing the detergent concentration in the solution. These data revealed the importance of the calcium in the structure of SLBs and provided new insights into the mechanism of protein insertion into these model membranes.
通过直接掺入支撑脂质双层（SLB）中来重构跨膜蛋白，是一种为膜蛋白的高分辨率原子力显微镜（AFM）分析提供合适样品的新方法。首批实验报道了蛋白成功掺入去污剂破坏的SLB中。在此，我们通过AFM分析了在钙存在的情况下膜蛋白的掺入情况，钙是对几种膜蛋白功能重要的二价阳离子。使用脂质相分离膜，我们首先表明钙能强烈稳定SLB，减少低临界胶束浓度去污剂（十二烷基-β-麦芽糖苷、十二烷基-β-硫代麦芽糖苷和N-十六烷基磷酰胆碱（Fos-Choline-16））的插入以及蛋白的进一步插入。然而，通过增加溶液中的去污剂浓度，在钙存在的情况下可恢复高蛋白插入率。这些数据揭示了钙在SLB结构中的重要性，并为蛋白插入这些模型膜的机制提供了新见解。
