Binding Protein Medicated Membrane Transport Systems

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The structure and function of the components of the binding protein mediated membrane transport system for ribose in E. coli will be probed by two main approaches. First, site-directed mutagenesis studies on the ribose binding protein will be continued in order to define the surfaces on the ribose binding protein which interact with the chemotaxis (trg) and the ribose transport receptors on the cytoplasmic membrane. Our recant studies of this type located one site near the "hinge" of the binding protein which eliminated chemotaxis when an Ile residue was changed to Arg. Further probing of this site and residues near it are planned as well as sites near the cleft of the binding protein which appear to affect transport. Second, overexpression, isolation, and characterization of the components of the membrane transport complex for ribose will be continued. Two of the three proteins in the complex have been purified, one of which has been shown to be homologous to a family of ATP-binding proteins including the eukaryotic multiple drug resistance proteins and the newly identified cystic fibrosis-related protein. Physical and biochemical studies will be performed on the purified transport protein, and attempts to overexpress the third will be continued. Once all three are produced, reconstitution of the transporter in vitro will be pursued. A reconstituted system, along with the information related to the surface of the binding protein which interacts with the transported, will allow future biochemical studies of the mechanism of transport, A related objective is to overexpress the repressor for the rbs operon, a molecule which was shown to be homologous both to a family of repressors and also to the family of binding proteins to which ribose binding protein belongs. This led to a model for the repressor mechanism which will be testable biochemically and structurally once significant amounts of the pure repressor are obtained.