TonB is a protein required for active transport of several ligand molecules across the outer membrane of E. coli, including iron-bearing siderophores and vitamin B12. The current model for TonB function is that it couples cytoplasmic membrane potential energy to conformational changes in the outer membrane receptors, which in turn results in transport of the ligands across the membrane. The goal of this project is to biochemically analyze TonB function within the context of this model. The relationship of TonB instability to its function will be investigated by immunoprecipitation of metabolically labeled TonB in pulse-chase experiments. Structural features of the unusual proline-rich regions of TonB will be analyzed for functional significance by creation of specific mutations using site-directed in vitro mutagenesis techniques. The role of potential membrane anchors at both the carboxyl and amino termini of the TonB proteins will be determined using membrane fractionation techniques combined with site-specific TonB mutants. This project will explore a structurally and functionally unusual and important membrane protein. The results will be of interest to protein biochemists as well as cell biologists, since general principles concerning structure-function relationships are likely to emerge. In addition, the information which will be generated concerning the structure and role of TonB in active transport of necessary nutrients may be useful in combatting pathogenic Gram- negative bacteria.
