Solving the structure with a view to understanding the function of membrane proteins remains one of the grand challenges in all of Biology. Given that a third of the human genome codes for membrane proteins, many of which serve signal transducing, structural and transport roles and are targets of disease causative and treatment agents, the health consequences of meeting the challenge are great. A multifaceted approach will be taken to advance our understanding of membrane protein function by producing structure grade crystals for use in macromolecular crystallography. Emphasis is placed on crystallization in lipidic mesophases by what is referred to as the 'in meso'or cubic phase method. This is proving to be a generally useful approach for membrane protein structure determination. With this method crystallization takes place in a membrane environment that is likely to be favored by the target membrane protein. We have built a unique, state-of-the-art robot that performs in meso crystallization in high-throughput fashion and that requires miniscule amounts of protein, lipid and precipitant. It will be used in the current application to produce 3-D crystals for the high-resolution structure determination of a select group of membrane proteins. The target group covers a broad range of membrane protein types including eukaryote and prokaryote, integral and peripheral, monomeric and multimeric, as well as protein-protein and protein-peptide complexes. Some of the target proteins will be produced in-house, some will be provided by individual collaborators, while others will be supplied through the NIH Structural Proteomics Initiative. In line with the NIH Structural Biology Road Map, and in parallel with the proposed structure study, effort will be devoted to improving crystallogenesis and to broadening the range of membrane protein targets that yield to structure determination. This will be achieved by implementing a synthesis program whereby lipids with desirable physico-chemical characteristics are produced for use in crystallization trials. The molecular mechanism of crystal nucleation and growth will be studied too with a view to more rational and successful crystallogenesis. Success in obtaining crystals, and ultimately the atomic structure of membrane proteins, will enhance our understanding of some of the most fundamental processes underlying cellular function that are integral to human health.
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