Since atomic structure is key to understanding mechanisms and to designing modulators, the long- term objective of this proposal is to establish the basis for understanding four functional classes of membrane proteins at this level of molecular structure and mechanism. In the aquaporin family (AQPs) specific goals include (1) determining the basis of selectivity using mutations in four AQPs whose structures have already determined in our laboratory, (2) expression and structure determination of a human aquaporin AQP4, with impact for drug design against collateral damage in stroke, and the malaria parasite aquaporin PfAQP, a new anti-malarial drug-target. A chemical 'tethering'approach will identify compounds that can be used to prove the principle of the malarial aquaporin as a drug target. Building on our landmark first molecular structure and mechanism of a transmembrane Ammonia transporter, AmtB, research is to establish the atomic basis for function using structural and functional analysis of mutant proteins, designed on the basis of our structure. A second goal in this family is determining the basis for AmtB regulation by GlnK. Structures of related family members that include the Rh subfamily present in eukaryotes will be sought. A third class is that of urea transporters where we seek to determine structures of a urea channel from the human pathogen Helicobacter pylorisince this channel is essential for pathogenicity in stomach ulcer. A second target is the urea transporter from P. aeruginosa. This target is homolgous to human urea transporters, particularly in the transmembrane regions, and thus presents an entry into this new membrane protein fold. Lastly we seek to refine conditions for expression in a membrane fraction, of a functional but simplified version of the neuronal nicotinic acetylcholine receptor obtained by heterologous expression of a unique single subunit, a7, that forms fully functional homopentameric AcChR channels. The techniques used in this research are evolving forefront methods for expression of membrane proteins, extraction and purification, crystallization, and structure determination of membrane proteins. Structural models will be tested and refined by mutational analysis coupled to structural and functional measurements on mutant proteins. The structural and functional analyses of native and mutant proteins will lead to procedures that can directly evaluate and/or establish their therapeutic advantage as drug targets, and provide a key to advancing their successful implementation as targets. While 40% of all targets for today's drugs are membrane proteins there is not yet a single atomic structure for any one such drug target membrane protein. The structures of these proteins will establish their mechanism of action and provide templates for drug discovery aimed at those of particular therapeutic advantage, and the essential elements to understand their selectivity.
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