Droplet Method for Bilayer Assays

描述(申请人提供)：膜蛋白对于了解人类健康和治疗疾病极其重要。它们占体内所有蛋白质的30%，参与调节信号转导和细胞内和细胞之间的分子运输，是当今大多数药物的靶标。为了正常发挥功能，膜蛋白必须嵌入脂膜层。对单个膜蛋白的良好控制研究需要创建一种人造膜来容纳蛋白质，但现有的制造人造膜的方法，如黑色类脂膜和具有固体支撑的膜，不能适应高通量技术，因为对于黑色类脂膜来说，这些膜相当脆弱，或者对于具有固体支撑的膜来说，获取途径有限。高通量方法允许同时检查大量化合物或大量相互作用，正越来越多地被用于研究影响人类健康的分子相互作用以及识别和测试药物。高通量筛查的重要性已在美国国立卫生研究院医学研究路线图中得到认可。NIH路线图的分子图书馆和成像部分旨在促进公共部门内新的小分子药物的开发。对这些小分子进行高通量筛选是拟议工作的一个组成部分。我们建议研究一种基于倒置乳状液的脂膜和膜蛋白的研究方法。用这种方法，我们可以容易地制备出体积小、表面积小的双层膜。我们期望该方法能方便地用于膜蛋白功能和影响膜蛋白的药物的高通量筛选。由于膜的尺寸较小，该技术将避免黑色类脂膜技术中典型的膜脆性问题。此外，由于膜是在两个倒置的乳液滴之间形成的，因此我们将避免在固体载体上使用人造膜时接触膜两侧的困难。由于所涉及的体积很小，我们可以最大限度地减少分析中使用的试剂的量，并可以在单一底物上快速进行大量的分析。我们建议通过以下方法来研究膜蛋白的反相乳化法：(1)通过表征双层膜相对于连续相、脂类组成和引入相的性能来研究反相乳状液中双层的形成，并通过渗透性研究来比较膜的性质；以及(2)使用插入的蛋白质或多肽来测试膜的性能。我们将把一个跨膜蛋白和一个带有跨膜多肽的自组装孔结合到界面双层中，并使用荧光染料来确定它们的存在和功能。公共卫生相关性：项目叙述跨膜蛋白是人体内一类非常重要的分子，也是当今大多数药物的靶标。这项研究为研究跨膜蛋白的功能开发了一种强大的新方法，这反过来应该会导致在治疗从感染到癌症到遗传性疾病的广泛疾病方面的改进。

DESCRIPTION (provided by applicant): Membrane proteins are extremely important to understanding human health and treating disease. They make up 30% of all proteins in the body, are involved in regulating signal transduction and molecular transport into and between cells, and are the target of most drugs available today. To function properly, membrane proteins must be embedded in a lipid membrane layer. Well-controlled studies of individual membrane proteins require creating an artificial membrane to hold the protein, but existing methods for producing artificial membranes, such as the black lipid membrane and membranes with solid support, cannot be adapted to high- throughput techniques because the membranes are quite delicate for black lipid membranes or have limited access for membranes with solid support. High-throughput methods, which allow examination of a large number of compounds or a large number of interactions simultaneously, are increasingly being used to study molecular interactions affecting human health and to identify and test drugs. The importance of high- throughput screening has been recognized in the NIH Roadmap for Medical Research. The Molecular Libraries and Imaging portion of the NIH Roadmap seeks to facilitate the development of new, small-molecule drugs within the public sector. High-throughput screening of these small molecules is an integral part of the proposed effort. We propose to investigate a method for studying lipid membranes and membrane proteins based on an inverted emulsion. With this method we can readily produce bilayer membranes with small volumes and small surface areas. We expect that this method will be easily adapted to high-throughput screening of membrane protein function and drugs influencing membrane proteins. Because of the small size of the membrane, this technique will avoid the membrane fragility issues typical of black lipid membrane techniques. Furthermore, because the membrane is formed between two inverted emulsion droplets, we will avoid the difficulties in accessing both sides of the membrane found with artificial membranes on solid support. With the small volumes involved, we can minimize the quantities of reagent used in assays and can perform very large numbers of assays rapidly on a single substrate. We propose to investigate the inverted emulsion method for membrane proteins by: (1) studying bilayer formation in an inverted emulsions by characterizing the bilayer performance with respect to the continuous phase and lipid composition and phase of introduction, and comparing the membrane properties with liposomes through permeability studies; and (2) testing membrane performance using inserted proteins or peptides. We will incorporate one transmembrane protein and one self- assembled pore with a transmembrane peptide into the interfacial bilayer and use fluorescent dyes to establish their presence and function. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Transmembrane proteins are a very important class of molecules in the human body and are the target of most drugs available today. This research develops a powerful new method for studying the function of transmembrane proteins that should in turn lead to improvements in treating a wide range of diseases ranging from infections to cancer to genetic disorders.