STRUCTURE/FUNCTION OF THE CHIP28 WATER CHANNEL

这项建议的目的是了解 哺乳动物的水通道CHIP28在双层中由其高分辨率的3- 维度(3-D)结构和结构调制由 电子结晶学。CHIP28，促进渗透水运输 跨红细胞膜和各种透水上皮细胞膜 作为对水有选择性的通道，不包括小离子和 溶质。我们的具体目标如下所示。 目的I.确定CHIP28分子在 双层的。我们将扩展关于投影结构的知识 通过计算从我们目前的低分辨率(14埃)地图中提取CHIP28 映射到逐渐更高的分辨率(8埃或更高)基于 未染色样品的图像和衍射图的分析 冰冻水合标本。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 多肽链的结构和对次生素的可能描述 结构。 目的II.确定CHIP28多肽链的三维结构 根据倾斜标本的数据进行重建。3-D密度贴图 将根据从以下地点收集的衍射图案和图像进行计算 冰冻水化的标本在显微镜下倾斜。这些地图生成 在逐步提高的分辨率下，将在3个维度上详细说明 多肽链的二级结构和三级折叠 遍历双层。最终目标是一种化学物质的假设 折叠包装水分运移调节模型 多肽链的。 目标三.研究抑制水的结构基础-- 汞化合物对CHIP28的迁移性能作为第一步 从CHIP28的调节机制来理解其功能 汞类药物抑制剂的结构作用将是 学习。最初，投影中的电子衍射图将是 以尽可能高的分辨率用于量化结构变化 当汞与已知的单个胞外CyS189残基结合时。 CHIP28属于所谓的MIP超家族通道蛋白，来自 可能具有相似结构/功能的各种生物 相互关联。因此，拟议的研究将有助于阐明BASIC 跨膜信号转导机制。CHIP28基因的选择性表达 和上皮细胞中的同源水通道被认为参与了 在液体吸收和/或分泌方面使其成为极其重要的 药理靶标。因此，高分辨率3-D结构将 可能会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.