Tools for Membrane Protein Structure Determination

描述（由申请人提供）： 滥用药物通过与膜受体、神经递质转运蛋白和配体门控离子通道（如5-HT 3受体）结合发挥作用。为了获得机制的理解和扩大治疗选择，结构信息是必不可少的，但膜蛋白一直难以获得所需的详细研究，通过X射线或电子晶体学的结晶状态。我们提出了一种新的方法来鉴定使用非洲爪蟾卵母细胞作为表达宿主容易形成二维（2D）晶体的膜蛋白。 一个非洲爪蟾卵母细胞可以产生许多倍的蛋白质，需要解决其结构，近原子分辨率的电子晶体学，但在同一时间是显着缺乏内源性膜蛋白。同样重要的是，大多数真核细胞膜蛋白可以在卵母细胞中表达，并且在某些情况下，这种表达导致体内自发的2D结晶。因此，显示出形成2D晶体的倾向的蛋白质是非常有前途的结构研究的目标。这项可行性研究的目的是开发新的方法来发现体内形成的2D晶体，或者在结晶不会自发发生的情况下鼓励原位结晶。 目的1：开发一种检测体内2D晶体自发形成的方法。膜蛋白的自发聚集与体内2D晶体自发形成的相关程度从未被研究过。由于聚集的荧光标记的膜蛋白可以很容易地检测到荧光显微镜，我们将确定是否荧光标记和筛选标点染色可以识别膜蛋白，容易形成2D晶体。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-HT 3受体。 目的2：建立一种新的方法来诱导非自发结晶的膜蛋白的原位二维结晶。卵母细胞膜中内源性膜蛋白的低背景可允许任何重组膜蛋白通过去除过量脂质而原位结晶。 传统上，洗涤剂用于此目的。然而，洗涤剂的作用难以控制，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在脱脂方案中取代去污剂。这种方法可以使大量膜蛋白适合2D结晶和结构测定。

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.