Membrane protein electrophoresis in lipid bilayers

描述(申请人提供)：提出了一种在保持功能的同时有效分离膜蛋白的新的电泳法。建议将液体、光滑、电中性的脂类双层膜作为电泳介质。拟议的分离将使膜蛋白保持在其自然类型的环境中，以产生分离的膜蛋白区域，然后以功能阵列的形式显示。这种分离将支持膜蛋白质组学、药物筛选和癌症诊断开发等应用。膜蛋白在人类健康中发挥着关键作用，因为它们直接引导细胞信号、识别和运输，然而分离膜蛋白用于研究的步骤昂贵、耗费人力且效率低下。今天分离蛋白质的方法依赖于水的溶解度。这项拟议的工作将通过研究三个基本性质来测试分离脂双层中膜蛋白的可行性。首先，我们将在原子转移自由基聚合法生长的超光滑聚丙烯酰胺膜上研究脂质双层的流动性和均匀性。聚丙烯酰胺膜在双层的底面提供了统一的亲水边界，上层将使用含水的缓冲液。其次，将研究5种特性良好的受体的电泳率：3种阿片受体和2种黑素皮质素受体。这5种受体都是医学上重要的G蛋白偶联受体。它们的配体是多肽，这些多肽将被罗丹明单独标记。第三，将通过罗丹明标记配体的单分子荧光光谱来研究这些膜蛋白的功能。R21的研究将关键地检验在支持的脂类双层中，电泳法分离功能性膜蛋白是否可行。如果可行，R01提案将解决从细胞裂解物中分离真正的膜蛋白混合物的问题。这项工作可能会对药物发现产生巨大影响，使涵盖许多受体的廉价筛选成为可能。

DESCRIPTION (provided by applicant): A new electrophoretic method is proposed for efficiently separating membrane proteins while retaining function. Fluid, smooth, electrically neutral lipid bilayers are proposed as the electrophoretic medium. The proposed separation would maintain the membrane proteins in their natural type of environment to produce separated zones of membrane proteins, which are then displayed as functional arrays. The separation would support such applications as membrane proteomics, drug screening and development of cancer diagnostics. Membrane proteins play key roles in human health, since they direct cellular signaling, recognition and transport, yet the steps for isolating membrane proteins for research are expensive, laborintensive and inefficient. Today's methods for separating proteins rely on water solubility. The proposed work will test the feasibility of separating membrane proteins in lipid bilayers by investigating 3 essential properties. First, the fluidity and uniformity of the lipid bilayer will be studied on ultrasmooth polyacrylamide films, grown by atom-transfer radical polymerization. The polyacrylamide film provides a uniform hydrophilic boundary on the underside of the bilayer, an aqueous buffer will be used on the upper side. Second, the electrophoretic mobilities of 5 well characterized receptors will be studied: the 3 opioid receptors and 2 melanocortin receptors. All 5 are medically important G-protein coupled receptors. Their ligands are peptides, which will be singly labeled with rhodamine. Third, the function of these membrane proteins will be studied by single-molecule fluorescence spectroscopy of the rhodamine labeled ligands. The R21 research will critically test the idea of whether the electrophoretic separation of functional membrane proteins is feasible in supported lipid bilayers. If feasible, an R01 proposal would address separations of real mixtures of membrane proteins from cell lysates. The work could have enormous impact on drug discovery, allowing inexpensive screening that encompasses many receptors.