HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

本研究的长期目标是了解水跨膜通道运输的功能机制。水通道蛋白（aquaporins，AQP）是存在于细菌、酵母、昆虫、植物、哺乳动物和两栖动物组织中的水通道蛋白家族，属于MIP（Major Intrinsic Protein，主要内在蛋白）超家族。 水通道蛋白对于正常细胞功能所需的水平衡的主动调节至关重要;例如，AQP 2的缺陷与诸如肾源性尿崩症的疾病有关。 AQP 1是水通道蛋白的一个子集，可以发现存在于来自器官的多种组织中，例如肾脏、胆囊、脾脏、肺、肠、内耳和眼睛。 这些通道被认为是水特异性的，在流体吸收和分泌期间将水运输穿过许多上皮和内皮细胞层。我们建议继续努力确定AQP 1的原子模型。 在目前的拨款年，我们已经获得了约3.5埃单位分辨率的AQP 1的投影图和约6埃单位的3D图。使用电子晶体学获得3.5埃单位的3D结构的集中努力面临着几个技术问题：高度倾斜样品的图像和衍射图案显示垂直于倾斜轴的非常有限的结构信息。 此外，目前使用电子晶体学确定的结构分辨率不足以清楚地观察水分子，这对于理解水通道蛋白的功能机制及其对水的特异性至关重要。 然而，获得用于X射线晶体学研究的膜蛋白的3D晶体的巨大困难一直是一个主要的绊脚石。 在过去的一年里，我们专注于3D结晶，并成功地获得了适合X射线晶体学研究的晶体。 天然日期集已被收集到约3埃单位分辨率，虽然晶体显示衍射斑点优于2.5埃单位分辨率。 我们现在正把主要精力集中在获得重原子衍生物上。在约3埃单位分辨率或更高分辨率下的水通道的结构将提供用于理解MIP超家族对水跨膜转运的调节的范例。原子模型将揭示通道的分子细节，包括调节其水运输特异性的机制。 它也将提供一个结构的基本原理的特异性的变化，从水甘油和蛋白质寡聚体状态的改变，从四聚体单体形式，观察AQPcic的双点突变体。 水通道的分子机制的知识可以提供深入了解蛋白质缺陷导致的疾病，如肾源性尿崩症的结构基础。

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 bacteria, yeast, insect, plant, mammalian and amphibian tissues and belong to the MIP (Major Intrinsic Protein) superfamily. Aquaporins are critical for the active regulation of water balance required for normal cell function; defects in AQP2, for example, have been related to diseases such as nephrogenic diabetes insipidus. AQP1 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, inner ear 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 continue our efforts in determining the atomic model of AQP1. In the current grant years, we have obtained the projection map of AQP1 at a resolution of about 3.5 Angstrom units resolution and a 3D map at approximately 6 Angstrom units. Concentrated efforts to obtain the 3D structure at 3.5 Angstrom units using electron crystallography were faced with several technical problems: images and diffraction patterns of highly tilted samples show very limited structural information normal to the tilt axis. In addition, the current resolution of structures determined using electron crystallography is not adequate to clearly observe water molecules which is crucial for understanding the aquaporin functional mechanisms and their specificity for water. However, the great difficulty in obtaining 3D crystals of membrane proteins for x-ray crystallographic studies has been a major stumbling block. In the last year, we have focused our efforts on 3D crystallization and were successful in obtaining crystals suitable for x-ray crystallographic studies. Native date sets have been collected to approximately 3 Angstrom units resolution, although the crystals show diffraction spots to better than 2.5 Angstrom units resolution. We are now focusing our major efforts on obtaining heavy atom derivatives. The structure of the water channel at approximately 3 Angstrom units resolution or higher would provide a paradigm for understanding the regulation of the transport of water across the membrane by the MIP superfamily. The atomic model will reveal the molecular details of the channel including the mechanism that regulates its specificity for water transport. It will also provide a structural rationale for the change in specificity, from water to glycerol and the alteration of the protein oligomeric state, from tetramer to monomeric form, observed in a double point mutant of AQPcic. The knowledge of the molecular mechanisms of water channels could provide insights into the structural basis of protein defects resulting in diseases such as nephrogenic diabetes insipidus.