HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

这项研究的长期目标是了解功能 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) 超级大家庭。水通道蛋白对正常细胞功能至关重要； 这些蛋白质与肾源性糖尿病等疾病有关。 尿崩症导致肾脏不能集中尿液 对加压素的反应。AQP-CHIP是水通道蛋白的一个子集，可以 存在于肾脏等器官的各种组织中， 胆、脾、肺、肠、眼。这些频道是 据信是特定于水的，将水输送到许多 液体吸收过程中的上皮层和内皮细胞层 分泌物。 我们建议用电子方法确定AQP-CHIP的分子结构 结晶学方法。我们将利用我们的成功在 膜蛋白与脂类重组，形成高度粘性 衍射率约为3.0埃的二维(2-D)晶体。我们 我已经获得了AQP-CHIP的投影图，分辨率约为3.5 埃分辨率。我们开始努力确定这三个人- 维度结构，最初为6埃分辨率，然后为3.5埃 埃斯特罗姆。6埃分辨率的结构将提供 关于跨膜螺旋的数量和组织的信息， 从投影图上看不出清晰的图像。这个中间体 解析结构也将被用作以下方面的起点和工具 以3.5分辨率解释后续的3-D重建 埃斯特罗姆。同时，我们将在以下方面作出重大努力 开发一种将产生更好质量的结晶协议 电子结晶学结构测定用晶体约 2.5埃；晶体的顺序和大小一直是主要限制 获取大于3.5埃数据的因素。 水通道的结构在3.5埃分辨率下将 为理解细胞周期调控机制提供了分子基础 水的跨膜运输。原子模型将揭示 调节其特异性的通道本身的分子细节 水路运输。这一谅解预计还将揭示 MIP超家族分子机制的一般原理 可以为蛋白质缺陷的结构基础提供洞察力。 对水通道蛋白的结构研究也可以提供线索 其他质膜通道的结构设计，这些通道不是 目前可用。我们建议的研究工作也将加强我们的 了解膜蛋白的二维结晶，这是 电子结晶学在膜材料中的广泛应用 蛋白质结构测定。

The long-term 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 belong to the MIP (Major Intrinsic Protein) superfamily. Aquaporins are critical for normal cell function; defects in these proteins have been related to diseases such as nephrogenic diabetes insipidus resulting in the failure of kidney to concentrate urine in response to vasopressin. AQP-CHIP is a subset of the aquaporins and can be found to exist in a variety of tissues from organs such as the kidney, gall bladder, spleen, lung, intestine and eyes. These channels are believed to be water specific, transporting water across a number of epithelial and endothelial cell layers during fluid absorption and secretion. We propose to determine the molecular structure of AQP-CHIP by electron crystallographic methods. We will exploit our success in the reconstitution of membrane protein with lipid, forming highly coherent two-dimensional (2-D) crystals that diffract to about 3.0 angstroms. We have obtained the projection map of AQP-CHIP at a resolution of about 3.5 angstroms resolution. We are beginning our effort to determine the three- dimensional structure, initially at 6 angstroms resolution and then at 3.5 angstroms. The structure of 6 angstroms resolution will provide information about the number and organization of transmembrane helices, which are not clearly resolved from the projection map. This intermediate resolution structure will also be used as a starting point and tool for interpreting subsequent 3-D reconstructions at a resolution of 3.5 angstroms. Simultaneously, we will devote significant effort toward developing a crystallization protocol that will yield better quality crystals for structure determination by electron crystallography to about 2.5 angstroms; the crystalline order and size have been major limiting factors in obtaining higher than 3.5 angstroms data. The structure of the water channel at 3.5 angstroms resolution will provide the molecular basis for understanding the regulation of the transport of water across membrane. The atomic model would reveal the molecular details of the channel itself that regulates its specificity for water transport. This understanding is expected to also reveal the general principle governing the molecular mechanism of the MIP superfamily and could provide insights into the structural basis of protein defects. The structural studies of water channel proteins could also provide clues to the structural design of other plasma membrane channels, which are not currently available. Our proposed research effort will also enhance our understanding of 2-D crystallization of membrane proteins, which is crucial for the widespread use of electron crystallography for membrane protein structure determination.