Binding-protein-dependent transport systems in bacteria are members of the ATP-binding cassette (ABC) superfamily and genetic defects in human homologs are responsible for several serious human diseases including cystic fibrosis. The maltose transport system of Escherichia coli is well characterized and is therefore an ideal model for the study of ABC transporters. A periplasmic maltose binding-protein (MBP) directs maltose to a membrane- associated transport complex containing two transmembrane- spanning proteins, Ma1F and Ma1G and two copies of a peripheral ATP-binding-protein, Ma1K. ATP hydrolysis by the multisubunit complex drives maltose transport. Our long term objective is to understand how the structural and mechanistic features of the maltose transport system result in active transport. As this mechanism is complex, our approach is to break the overall reaction down into smaller partial reactions that can be studied in isolation. In this proposal, we will elucidate the molecular mechanism of maltose transport by developing assays for conformational changes within the multi-subunit transport complex that contribute to the overall translocation event. Given that conformational signals must pass between the hydrophobic domains and the ATP-binding domains, we will focus primarily on this interface. We have available highly purified preparations in which it is possible to monitor the dynamics of the transport complex using biochemical and biophysical techniques. These purified preparations will also be used in collaborative efforts to obtain a high resolution structure of the multi-subunit complex.
