HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

这项研究的长期目标是理解功能 水在膜通道中的传输机制。水通道蛋白 水通道蛋白(AQP)是一类水通道蛋白，存在于植物、哺乳动物和 两栖动物组织，属于MIP(主要固有蛋白)超 一家人。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，如 肾源性尿崩症。AQP-1是水通道蛋白的一个亚家族， 可以发现存在于器官的各种组织中，如 肾、胆、脾、肺、肠、眼。这些频道是 据信专门将水分子输送到许多 液体吸收过程中的上皮层和内皮细胞层 分泌物。水通道蛋白的另一个亚家族，AQP-7，最近被 被鉴定并被发现专用于尿素的运输和 甘油和水。 我们建议用电子方法确定AQP-1的原子结构 结晶学方法。我们已经在以下位置获得了AQP-1的投影图 约3.5埃的分辨率和三维(3-D)地图 分辨率约为6埃。我们正在继续努力确定 3-D地图需要大约3.5埃的分辨率才能获得原子 这种膜通道蛋白的模型。在这一努力的同时，我们 将致力于从牛附睾液中提纯AQP-7 以获得足够进行结晶试验的量。 AQP-1和AQP-7的结构测定可望得到 对国际货币基金组织作用机制的一般原则的见解 在其中的水道中，我们的结构知识如果非常有限。这个 这两个不同的水通道亚家族的分子结构 将提供分子基础，以了解它们对 水、甘油和尿素的跨细胞膜运输。这样的一种 预计理解将揭示出管理 MIP超家族的分子机制，并可提供洞察力 与疾病相关的蛋白质缺陷的结构基础。这个 对这些水道的结构研究也有望提供 关于离子通道分子设计的线索 结构信息目前不可用。我们建议的研究 努力也将加强我们对二维(2-D)的理解 膜蛋白的结晶对于广泛的 电子结晶学在膜蛋白结构研究中的应用 决心。

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.