X-ray diffraction data is the main source of 3-dimensional structure information at atomic resolution for proteins. While a great deal of information is potentially available from this technique, its application requires the ability to prepare crystals of size and order suitable for X-ray analysis. This has proved especially difficult for membrane proteins. Complex n (succinate:ubiquinol oxidoreductase) is a membrane protein complex that funnels electrons from succinate into the mitochondrial respiratory chain. The respiratory chain is responsible for biological oxidation and for conservation of the energy released in the form of a proton electrochemical potential gradient across the mitochondrial inner membrane. Energy from this gradient is then used to synthesize ATP or to do work by transporting substances across the membrane. A number of mitochondrial myopathies and CNS disorders have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in complex n. In addition several subunits of Complex n have tumor suppressor activity, and defects in these subunits lead to paraganglioma. Complex n has been proposed to play a role in induction of apoptosis, which may be responsible for the tumor-suppressor activity. Structures have recently become available for a number of bacterial homologs of Complex n, however no structure is available for a vertebrate complex n, or any mitochondrial complex n for that matter. In previous work we were occasionally able to crystallize avian Complex II with unit cell edges 69 x 84 x 290 A, in the spacegroup P2]2i2i. Now we have greatly improved the reproducibility and quality of the crystals and worked out a freezing protocol that Allows cryogenic data collection at up to 2.0 A. The structure has been solved and is being refined against a 2.2 A dataset from a crystal of protein treated with oxaloacetate. It is proposed to (a) Further optimize the cryoprotection protocol to minimize mosaicity and freezing damage (b) Collect high-reolution diffraction data from crystals of the as-prepared protein as well as protein cocrystallized with a number of medically or mechanistically relevant ligands at the substrate and ubiquinone binding sites, (c) Complete the refinement of the native structure, and use it to solve the structures with various ligands. Also (d), we want to spend some time to understand how the crystallization and cryoprotection procedures work in order to apply the knowledge to crystallographic studies of other membrane proteins. Finally (e) we want to crystallize a related complex n from an organism amenable to site-directed mutagenesis.
