Binding-protein-dependent transport systems in bacteria are members of the ATP-binding cassette (ABC) superfamily. ABC transporters secrete virulence factors in bacteria and contribute to the drug resistance of several pathogens. Genetic defects in human homologs are responsible for over a dozen serious human diseases including cystic fibrosis, adrenoleukodystrophy and cholestasis. The maltose transport system of E. coli is well characterized and is therefore an ideal model for the study of ABC transporters. A periplasmic MBP directs maltose to a membrane-associated transport complex containing two transrnembrane-spanning proteins, MalF and MalG, and two copies of a peripheral ATP-binding-protein, MaIK. ATP hydrolysis by the multisubunit complex drives maltose transport. The applicant's 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, the approach is to stabilize intermediates in the pathway and characterize them using biochemical and biophysical approaches. In this project, the investigators will use site-directed spin labeling and EPR, along with other complementary techniques, to define the movements that occur in moving from one intermediate to the next. The purified preparations will also be used in collaborative efforts to obtain high-resolution structures of the multi-subunit complex in different conformational states.
