The superfamily of translocators, traffic ATPases (or ABC proteins), the cystic fibrosis transmembrane conductance regulator (CFTR), the P- glycoprotein of multidrug resistance (MDR), and bacterial periplasmic permeases. Multidrug resistance is one of the major problems in cancer chemotherapy an cystic fibrosis is the most common recessive caucasian disease. Periplasmic permeases have been extensively studied and provide a good model system for understanding the mechanism of action of the medically relevant eukaryotic members of the superfamily. One such permease, the histidine permease, has been characterized in detail. As is true for traffic ATPases in general, the histidine permease is composed of two hydrophobic domains that are integral parts of the membrane, and of two hydrophilic domains that are also inserted into the membrane and bind ATP. Hydrolysis of ATP is used as the energy source. Since CFTR appears to be a channel, it is important to determine whether prokaryotic systems also function as channels. This would be an entirely novel concept for the prokaryotic systems. From the known structure of the membrane-bound complex, it is indeed possible that the hydrophobic domains of periplasmic permeases form a channel through which the substrate crosses the membrane, with ATP hydrolysis resulting in the necessary conformational changes. A characteristic peculiar to periplasmic permeases is the presence of a receptor that concentrates the substrate at the external surface of the membrane-bound complex. The receptor sends a signal to the membrane-bound complex, resulting in ATP hydrolysis and translocation. Among the tools that will be used in this study are several reconstituted systems and several measurable enzymatic activities that permit in vitro assays of function. The activity of traffic ATPases as channels will be investigated in lipid bilayers. The mechanism of signaling between the soluble receptor and the membrane-bound complex, in particular the energy- coupling component, will be studied by the use of biochemical reactions that distinguish between different conformations of proteins, such as limited proteolysis and covalent labeling, and by genetic analysis through the isolation of mutants with altered signaling processes. Similar biochemical and genetic procedures will be used to study the architecture of the membrane-bound complex. In addition, the components of the membrane-bound complex will be purified and characterized individually. Both two- and three-dimensional crystallography will be attempted to understand the structures of both the complex and the subunits. In addition to solving basic questions related to the mechanism of action of permeases in general, the study of this prokaryotic model system will help the efforts of eukaryotic researchers towards a solution of the medical problems related to multidrug resistance, cystic fibrosis, malarial parasite containment, and others.
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