Membrane protein electrophoresis in lipid bilayers

描述（由申请人提供）：提出了一种新的电泳方法，用于有效分离膜蛋白，同时保留功能。流体，光滑，电中性的脂质双层被提议作为电泳介质。所提出的分离将使膜蛋白保持在其天然类型的环境中，以产生分离的膜蛋白区，然后将其显示为功能阵列。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起着关键作用，因为它们指导细胞信号传导，识别和运输，但分离膜蛋白用于研究的步骤昂贵，劳动密集且效率低下。今天的分离蛋白质的方法依赖于水溶性。这项工作将通过研究3个基本性质来测试在脂质双层中分离膜蛋白的可行性。首先，超光滑聚丙烯酰胺薄膜，原子转移自由基聚合生长的脂质双层的流动性和均匀性将进行研究。聚丙烯酰胺膜在双层的下侧提供均匀的亲水性边界，在上侧使用水性缓冲液。第二，将研究5种表征良好的受体的电泳迁移率：3种阿片受体和2种黑皮质素受体。所有5种都是医学上重要的G蛋白偶联受体。它们的配体是肽，将单独用罗丹明标记。第三，这些膜蛋白的功能将研究通过单分子荧光光谱的罗丹明标记的配体。R21研究将严格测试功能性膜蛋白的电泳分离在支持的脂质双层中是否可行的想法。如果可行的话，R01提案将解决从细胞裂解物中分离膜蛋白的真实的混合物。这项工作可能会对药物发现产生巨大影响，允许廉价筛选包括许多受体。

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.