HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

水通道高分辨率电子显微镜

• 批准号: 6325878
• 负责人: Bing K. Jap
• 金额: $ 22.3万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325878
• 关键词: crystallization; electron microscopy; protein structure; water channel

The long range objective of this research is to understand the functional mechanism of water transport across membrane channels. The aquaporins (aqp) are a family of water channel proteins found in plant, mammalian and amphibian tissues and belongs to the MIP (Major Intrinsic Protein) super family. Aquaporins are therefore critical for normal cell function, and defects in these proteins have been related to diseases such as nephrogenic diabetes insipidus. AQP-1 is a sub-family of the aquaporins, and can be found to exist in a variety of tissues from organs such as kidney, gall bladder, spleen, lung, intestine and eyes. These channels are believed to specifically transport water molecules across a number of epithelial and endothelial cell layers during fluid absorption and secretion. Another sub-family of aquaporins, AQP-7, has recently been identified and has been found to be specific for the transport of urea and glycerol in addition to water. We propose to determine the atomic structure of AQP-1 by electron crystallographic methods. We have obtained the projection map of AQP-1 at a resolution of about 3.5 Angstroms and a three-dimensional (3-D) map about 6 Angstroms resolution. We are continuing in our effort to determine the 3-D map to about 3.5 Angstroms resolution needed to obtain an atomic model of this membrane channel protein. In parallel to this effort, we will devote significant effort to purify AQP-7 from bovine epididymis in order to obtain quantities sufficient for crystallization trials. The structure determination of AQP-1 and AQP-7 can be expected to yield insights into the general principles of the functional mechanisms of the water channels in which our structural knowledge if very limited. The molecular structures of these two different sub-families of water channels will provide the molecular basis for understanding their regulation of the transport of water, glycerol and urea across cell membranes. Such an understanding is expected to reveal the general principles governing the molecular mechanisms of the MIP super family, and could provide insights into the structural basis of disease-related protein defects. The structural studies of these water channels can also be expected to provide clues concerning the molecular design of ion channels for which direct structural information is currently unavailable. Our proposed research effort will also enhance our understanding of the two-dimensional (2-D) crystallization of membrane proteins which is crucial to the widespread use of electron crystallography for membrane protein structure determination.

本研究的长期目标是了解 水通过膜通道的运输机制。所述水通道蛋白 (aqp)是在植物、哺乳动物和哺乳动物中发现的水通道蛋白家族， 两栖动物组织，属于MIP（主要内在蛋白）超级 家人因此，水通道蛋白对正常细胞功能至关重要， 这些蛋白质的缺陷与疾病有关， 肾源性尿崩症AQP-1是水通道蛋白的一个亚家族， 并且可以发现存在于来自器官的多种组织中， 肾、胆、脾、肺、肠和眼。这些信道 据信它能将水分子运送到 上皮和内皮细胞层， 分泌物。水通道蛋白的另一个亚家族，AQP-7，最近被发现。 已鉴定并发现其对尿素的运输具有特异性， 除了水之外还有甘油。 我们建议用电子能谱测定AQP-1的原子结构 晶体学方法我们已取得AQP-1的投影图， 分辨率约为3.5埃， 6埃分辨率。我们正在继续努力确定 获得原子分辨率所需的约3.5埃分辨率的3-D映射 这种膜通道蛋白的模型。与此同时，我们 将致力于从牛附睾中纯化AQP-7， 以获得足够用于结晶试验的量。 AQP-1和AQP-7的结构测定有望产生 的功能机制的一般原则的见解， 我们对水道结构的了解非常有限。的 这两种不同的水通道亚族的分子结构 将提供分子基础，以了解他们的调控， 水、甘油和尿素跨细胞膜的运输。这样的 理解预计将揭示管理的一般原则， MIP超家族的分子机制，并可以提供见解 疾病相关蛋白质缺陷的结构基础。的 这些水道的结构研究也有望提供 关于离子通道的分子设计的线索， 结构信息目前不可用。我们提出的研究 这一努力也将提高我们对二维（2-D） 膜蛋白的结晶，这是至关重要的广泛 电子晶体学在膜蛋白结构中的应用 保持战略定力