Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.
Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.
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DOI：
10.1371/journal.pone.0024488
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Nollert P
中科院分区：
文献类型：
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作者：
Wallace E;Dranow D;Laible PD;Christensen J;Nollert P
膜蛋白在富含两亲性物质（如脂质立方相）中的结晶是许多结构生物学实验室建立的一种方法。该方法采用的标准程序需要通过将脂质（例如单油质）与洗涤剂溶解膜蛋白的溶液混合来产生高粘性的脂质物质。这种制备通常使用专门的混合工具进行，允许处理高粘性材料，同时最大限度地减少死体积，以节省宝贵的膜蛋白样品。脂质与膜蛋白最初混合时发生的过程尚不清楚。在这里，我们表明脂质相的形成和膜蛋白进入这些材料的结合可以通过实验分离。具体来说，我们研究了不同初始单油质脂相状态对球形红杆菌彩色光合反应中心结晶行为的影响。我们发现，洗涤剂溶解的光合反应中心自发地插入并浓缩在脂质基质中，没有任何混合，并且脂质物质的初始相态与生产结晶无关。在脂质物质表面的薄层中，膜蛋白的大量原位富集达到了否则无法达到的浓度水平。这些结果具有重要的实际应用价值，因此我们提出了一种在富含两亲性物质中进行膜蛋白结晶的简化方案，消除了在脂质立方相中制备结晶实验的任何专门的混合工具。此外，由于在单次结晶实验中采样膜蛋白浓度梯度，该结晶技术更加稳健，并提高了识别生产结晶参数的效率。最后，我们提供了一个模型来解释膜蛋白从溶液中通过门脉板层相进入脂质相的过程。
The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase.
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