STRUCTURE/FUNCTION OF THE CHIP28 WATER CHANNEL

本提案的目的是了解 哺乳动物水通道CHIP 28在双层从其高分辨率3- 三维（3-D）结构和结构调制， 电子晶体学促进渗透水运输的CHIP 28 穿过红细胞和各种透水性上皮细胞膜 作为对排除小离子的水有选择性的通道， 溶质。我们的具体目标如下。 AIM岛确定CHIP 28分子在细胞中的投影结构。 双层。 我们将扩展的投影结构的知识， CHIP 28从我们目前的低分辨率（14埃）地图通过计算 映射到逐渐更高的分辨率（8埃或更高）， 分析从未染色的样品中收集的图像和衍射图案 冷冻水化标本这些地图将提供更精细的定义， 分子包络、分子间堆积和路径约束 多肽链和可能的描述元素的二级 结构 AIM II.确定CHIP 28多肽链的三维结构 根据倾斜标本的数据进行重建。三维密度图 将根据衍射图案和图像进行计算， 在显微镜下倾斜的冷冻水化标本。这些地图生成 在逐步更高的分辨率将详细说明在3维， 多肽链的二级结构和三级折叠， 穿过双层。最终目标是假设一种化学物质 从折叠包装看水运调控模式 的多肽链。 AIM III.检查抑制水的结构基础- CHIP 28对汞化合物的迁移性能。作为第一步 为了从CHIP 28的调制中了解其功能， 汞药理学抑制剂的结构作用将是 研究了电子衍射图案，最初，在投影中将是 以尽可能高的分辨率用于定量结构变化 在汞结合到已知的单个细胞外CYS 189残基后。 CHIP 28属于所谓的MIP通道蛋白超家族， 可能具有相似结构/功能的不同生物体 相互关联因此，拟议的研究将有助于阐明基本的 跨膜信号传导机制。CHIP 28的选择性表达 以及上皮细胞中的同源水通道 在液体吸收和/或分泌中， 药理学靶点因此，高分辨率的3D结构将 可能对基于结构的药物设计产生影响。

The objective of this proposal is to understand the function of the mammalian water channel CHIP28 in the bilayer from its high-resolution 3- dimensional (3-D) structure and structural modulations determined by electron crystallography. CHIP28 which facilitates osmotic water transport across erythrocyte and various water-permeable epithelial cell membranes functions as a channel selective to water excluding small ions and solutes. Our specific aims are listed below. AIM I. Determine the projected structure of the CHIP28 molecule in the bilayer. We will extend the knowledge of the projection structure of CHIP28 from our current low-resolution (14 Angstroms) map by computing maps to progressively higher resolution (8 Angstroms and better) based on analysis of images and diffraction patterns collected from unstained frozen-hydrated specimens. These maps will provide finer definitions of the molecular envelope, intermolecular packing and constraints on the path of the polypeptide chain and possible description of elements of secondary structure. AIM II. Determine the 3-D structure of the polypeptide chain of CHIP28 from reconstructions based on data from tilted specimens. 3-D density maps will be calculated based on diffraction patterns and images collected from frozen-hydrated specimens tilted in the microscope. These maps generated at progressively higher resolution will elaborate in 3 dimensions the secondary structure and tertiary folding of the polypeptide chain as it traverses the bilayer. The ultimate goal is the postulation of a chemical model for the regulation of water transport from the folding and packing of the polypeptide chain. AIM III. Examine the structural basis for the inhibition of the water- transporting property of CHIP28 by mercurial compounds. As a first step towards understanding function of CHIP28 from its modulations, the structural role of the mercurial pharmacological inhibitor will be studied. Electron diffraction patterns, initially, in projection will be used at the highest possible resolution to quantitate structure changes upon mercurial binding to the known single extracellular CYS189 residue. CHIP28 belongs to the so-called MIP super-family of channel proteins from diverse organisms which likely share similar structure/function correlates. Thus the proposed research will add to elucidation of basic mechanisms of transmembrane signaling. The selective expression of CHIP28 and homologous water channels in epithelial cells believed to be involved in fluid absorption and/or secretion makes it an extremely important pharmacological target. Thus high-resolution 3-D structure will potentially have impact on structure-based drug design.