HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

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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.