X-ray diffraction data is by far 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. Cytochrome reductase (the cytochrome bc1 complex) is a membrane protein complex which makes up the middle segment of 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. In recent years a number of mitochondrial myopathies have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in cytochrome reductase. We have recently developed a procedure for preparing crystals of bovine heart mitochondrial cytochrome reductase. In addition to needle shaped crystals, which have been reported by other workers, we have obtained crystals in the shape of hexagonal bipyramids. These can be produced with a good rate of success by using a seeding technique. It seems likely that these crystal forms, when conditions for growth are optimized and crystals are larger, will prove suitable for X-ray diffraction analysis and eventually lead to a 3-dimensional structure of the complex at atomic resolution. The purpose of the research proposed here is to find conditions for growth of such suitable crystals. The effort will be applied to (1) improving the homogeneity of the purified enzyme, (2) choosing additives such as specific inhibitors or substrates that may stabilize one particular conformation of the enzyme, and (3) optimizing conditions of crystallization (temperature, ionic strength, protein concentration, precipitant species and concentration, detergent, and specific ions). Results will be monitored by size of the crystals obtained and by X-ray diffraction intensity and resolution. Once suitable crystals are available the effort will shift to collection of diffraction data and obtaining suitable heavy atom derivatives for subsequent structure determination.
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