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.
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