This investigation will focus on two membrane proteins involved in transfer of charge solutes across biological membranes. The adenine nucleotide carrier protein exchanges ATP and ADP across the mitochondrial inner membrane, allowing ATP generated in the mitochondrial matrix to be utilized in the cytoplasm. The proton translocating ATPase of plant plasma membranes maintains a membrane potential and pH gradient across the plasma membrane, which is important for nutrient uptake and in maintaining turgor pressure within the cell. While some biochemical characterization of these proteins will be included, the principle goal is to prepare membrane crystals of the proteins suitable for electron crystallography and to use these crystals to obtain a three dimensional structure for each protein. The effort to prepare highly ordered membrane crystals can be divided into three stages . The first is to find a purification procedure that produces the protein at a high level of purity and in a highly native state. Low levels of impurities, or slight alteration of some of the molecules could prevent crystallization or reduce the order in crystals. The second stage is to reconstitute the protein into membranes at high protein to lipid ratios. While routine procedures are available for incorporating both proteins into lipsomes to demonstrate transport, it remains to be seen whether they can be incorporated into membranes at high enough levels to make crystallization likely. The third stage is induction of crystallization in the membrane-embedded proteins. This depends on the interactions between the protein molecules, which in turn depend on pH relative to the isoelectric point of the protein, ionic strength, and specific factors that can ensure a single conformation for all protein molecules in the reconstituted bilayer. Conformational homogeneity may also be very important. Because each of the proteins exist in two different conformational states, substrate analog inhibitors that favor one state or the other will be added in most experiments to eliminate this source of heterogeneity.
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