EXCEED THE SPACE PROVIDED. A substantial fraction of the genes in any genome code for proteins whose functional role is carried out only after insertion into a lipid bilayer. While these proteins are amenable to structural studies by x-ray diffraction. they have tended to be considerably more difficult to crystallize than conventional soluble proteins, and structural studies of these vital proteins are lagging far behind. Over the last decade a number of respiratory chain membrane protein complexes have been solved structurally, including the cytochrome bcl complex (ubiquinol:cytochrome c oxidoreductase). This protein complex comprises the middle section of the mitochondrial respiratory chain, and defects lead to various myopathies and neurodegenerative diseases. In addition this protein is an important target of inhibitor design for crop protection agents and drugs for treatment of malaria or secondary infections in AIDS patients. The function has been extensively probed by site-directed and inhibitor-resistant mutations, mainly in Rhodobacter and yeast. The NIH project being proposed here for renewal was responsible for three structures of the avian mitochondrial bcl complex with different substrates and inhibitors bound. Now that the initial rush to obtain and deposit the structures is over, we propose a three-pronged approach to exploit the information and technology we have obtained in crystallization of these proteins. (1) Continued structure refinement, data mining, and functional interpretation based on the tremendous amount of information available even in our current 2.5-3 ,&, structures of this huge protein complex (2) Further improvement of the crystals through (2a)improved purification and crystallization conditions, better cryogenic technique, and new crystal forms (2b)Studying the crystallization process itself, not only to allow better refinement of crystallization conditions and control of the crystallization process for improving crystals but to understand the process and if possible develop some general principles that can be used for membrane protein crystal structures. (3) Location and characterization of the quinone/quinol bound at the Qp site. (4) Structure determinations with inhibitors of functional interest or of clinical or commercial importance bound to the quinone binding sites, as a test of mechanistic models and to understand and combat drug resistance when it arises. (5) Structural studies of functionally relevant mutant bacterial and yeast complexes. PERFORMANCE SITE ========================================Section End===========================================
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