Tools for Membrane Protein Structure Determination

描述(由申请人提供)： 滥用药物通过与膜受体、神经递质转运体和配体门控离子通道(如5-HT3-受体)结合而发挥作用。为了获得机理上的了解和扩大治疗选择，结构信息是必不可少的，但膜蛋白在晶体状态下很难获得，这是通过X射线或电子结晶学进行详细研究所需的。我们提出了一种新的方法来识别容易形成二维(2D)晶体的膜蛋白，该方法以非洲爪哇卵母细胞为表达宿主。一个非洲爪哇卵母细胞可以产生数倍于电子结晶学解析其结构到接近原子分辨率所需的蛋白质，但同时明显缺乏内源性膜蛋白。同样重要的是，大多数真核膜蛋白都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，具有形成2D-晶体倾向的蛋白质是结构研究的极有希望的目标。这项可行性研究的目的是开发新的方法来寻找在体内形成的2D晶体，或者在不自发结晶的情况下鼓励原位结晶。 目的1：建立一种检测体内2D晶体自发形成的方法。膜蛋白的自发聚集在多大程度上与体内2D晶体的自发形成相关还从未被研究过。由于荧光显微镜可以很容易地检测到荧光标记的膜蛋白的聚集，我们将确定荧光标记和点状染色筛选是否能够识别容易形成2D晶体的膜蛋白。为了实现这一目标，我们将使用荧光标记的“阳性对照”(连接蛋白32和50)和“未知”，如5-HT3受体。 目的2：建立一种新的诱导非自发结晶的膜蛋白原位2D结晶的方法。卵母细胞膜中内源性膜蛋白的低本底可以使任何重组膜蛋白通过去除多余的脂肪而原位结晶。传统上，洗涤剂是用于这一目的的。然而，洗涤剂的作用很难控制，它只有少量的膜可用。因此，我们将测试脂结合蛋白是否可以在脱脂方案中取代洗涤剂。这种方法可能使大量的膜蛋白易于2D结晶和结构确定。

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.