Droplet Method for Bilayer Assays

双层测定的液滴法

基本信息

批准号：
7835796
负责人：
GREGORY W FARIS
金额：
$ 22.55万
依托单位：
SRI INTERNATIONAL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-08 至 2012-04-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：膜蛋白对于理解人类健康和治疗疾病极为重要。它们占体内所有蛋白质的30％，参与调节信号转导和细胞之间和分子的分子转运，并且是当今大多数药物的靶标。为了正常工作，必须将膜蛋白嵌入脂质膜层中。对单个膜蛋白的控制良好的研究需要创建一种人造膜以容纳蛋白质，但是现有的生产人造膜的方法，例如黑脂质膜和具有稳固支撑的膜，不能适应高吞吐量技术，因为对于黑色脂质膜或限制了象征的薄膜，膜非常精致。高通量方法允许同时研究大量化合物或大量相互作用，越来越多地用于研究影响人类健康并识别和测试药物的分子相互作用。在NIH路线图中，高吞吐量筛查的重要性已被确认。 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。这些小分子的高通量筛选是拟议工作的组成部分。我们建议研究一种基于倒乳液的脂质膜和膜蛋白的方法。通过这种方法，我们可以轻松地生产具有少量体积和较小表面积的双层膜。我们预计该方法将很容易适应膜蛋白功能和影响膜蛋白的药物的高通量筛选。由于膜的尺寸很小，因此该技术将避免膜脆性问题的典型黑脂质膜技术。此外，由于膜是在两个倒乳液的液滴之间形成的，因此我们将避免在使用人造膜上找到具有固体支撑的膜的两侧的困难。随着涉及的少量，我们可以最大程度地减少测定中使用的试剂量，并且可以在单个底物上迅速执行大量测定。我们建议通过以下方式研究膜蛋白的倒乳液方法：（1）通过表征双层乳液中的双层形成，通过表征双层性能在连续相和脂质组成和引入相位，以及将膜性质与脂质体进行比较。（2）使用插入的蛋白质或肽测试膜性能。我们将将一种跨膜蛋白和一个自组装的孔与跨膜肽结合到界面双层中，并使用荧光染料来确定其存在和功能。公共卫生相关性：项目叙事跨膜蛋白是人体中非常重要的分子类，是当今大多数药物的靶标。这项研究开发了一种研究跨膜蛋白功能的有力新方法，反过来又应改善治疗从感染到癌症再到遗传疾病的广泛疾病。