Tools for Membrane Protein Structure Determination

膜蛋白结构测定工具

基本信息

批准号：
6771889
负责人：
VINZENZ UNGER
金额：
$ 16.16万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-07-01 至 2005-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Drugs of abuse act through binding to membrane receptors, neurotransmitter transporters, and ligand-gated ion channels such as the 5-HT3-receptor. To gain mechanistic understanding and expand therapeutic options, structural information is essential, but membrane proteins have been difficult to obtain in the crystalline state needed for detailed study by x-ray or electron crystallography. We propose a novel approach to identify membrane proteins that readily form two-dimensional (2D) crystals using Xenopus oocytes as expression host. A single Xenopus oocyte can produce many times the amount of protein needed to solve its structure to near atomic resolution by electron crystallography, yet at the same time is remarkably deficient in endogenous membrane proteins. Equally important, most eukaryotic membrane proteins can be expressed in oocytes, and in some cases such expression leads to spontaneous 2D-crystallization in vivo. Proteins that thus show propensity to form 2D-crystals are highly promising targets for structural studies. This feasibility study aims, at developing new approaches to find 2D-crystals that form in vivo or to encourage in situ crystallization in cases where crystallization does not occur spontaneously. Aim 1: Develop an assay to detect spontaneous formation of 2D-crystals in vivo. The extent to which spontaneous clustering of membrane proteins correlates with spontaneous formation of 2D-crystals in vivo has never been investigated. Since clustering of fluorescently labeled membrane proteins can easily be detected by fluorescence microscopy, we will determine whether fluorescence labeling and screening for punctuate staining can identify membrane proteins that readily form 2D-crystals. To achieve this goal, we will use fluorescently labeled "positive controls" (connexin32 and 50) and "unknowns" such as the 5-HT3-receptor. Aim 2: Develop a novel method to induce in situ 2D-crystallization of membrane proteins that do no crystallize spontaneously. The low background of endogenous membrane proteins in oocyte membranes may allow any recombinant membrane protein to be crystallized in situ by removing excess lipid. Traditionally, detergents are used for this purpose. However, the action of detergents is difficult to control it only small amounts of membranes are available. Therefore, we will test whether lipid-binding proteins can replace detergents in delipidation protocols. This approach may make a large number of membrane proteins amenable to 2D-crystallization and structure determination.

描述（由申请人提供）：通过与膜受体，神经递质转运蛋白和配体门控离子通道（如5-HT3受体受体）结合，滥用药物。为了获得机械理解和扩展治疗选择，结构信息至关重要，但是在X射线或电子晶体学详细研究所需的晶体状态下，很难获得膜蛋白。我们提出了一种新的方法来鉴定膜蛋白，该膜蛋白很容易使用爪蟾卵母细胞作为表达宿主形成二维（2D）晶体。单个爪蟾卵母细胞可以生产许多蛋白质，以通过电子晶体学求解其结构所需的蛋白质量，同时在内源性膜蛋白中非常缺乏。同样重要的是，大多数真核膜蛋白可以在卵母细胞中表达，在某些情况下，这种表达会导致体内自发的2D-结晶。因此显示出形成2D-晶体倾向的蛋白质是结构研究的高度有希望的靶标。这项可行性研究的目的是开发新的方法，以发现体内形成的2D晶体或在不自发结晶发生的情况下鼓励原位结晶。目标1：开发一种检测体内2D-晶体的自发形成的测定。膜蛋白的自发聚类的多大程度与体内2D-晶体的自发形成相关。由于可以通过荧光显微镜很容易检测到荧光标记的膜蛋白的聚类，因此我们将确定荧光标记和点点染色的筛选是否可以鉴定易于形成2D-晶体的膜蛋白。为了实现此目标，我们将使用荧光标记的“阳性对照”（Connexin32和50）和“未知数”，例如5-HT3受体。目标2：开发一种新的方法来诱导不会自发结晶的膜蛋白的原位2D结晶。卵母细胞膜中内源性膜蛋白的低背景可能使任何重组膜蛋白通过去除过量的脂质而在原位结晶。传统上，洗涤剂用于此目的。但是，清洁剂的作用很难控制它，只有少量的膜可用。因此，我们将测试脂质结合蛋白是否可以替代删除方案中的洗涤剂。这种方法可能会使大量的膜蛋白适合2D结晶和结构测定。