USING CHIMERAS TO EVALUATE CROSS-TALK, ENERGY TRANSFER, AND PROTEIN-PROTEIN INTERACTIONS IN THE TONB AND TOLA SYSTEMS
Date of Award
Doctor of Philosophy (Ph.D.)
The cytoplasmic membrane (CM) protein TonB energizes transport of ferric siderophores and group B colicins across the gram-negative outer membrane, and confers sensitivity to certain bacteriophages. These functions depend upon heteromultimeric protein complexes that couple TonB to the ion electrochemical potential of the CM. This is most efficient when TonB interacts with the ExbB/ExbD complexes; however, the ExbB/ExbD paralogues TolQ/TolR can also support TonB-dependent processes, albeit less efficiently. Strains bearing specific deletions of exbB, exbD, tolQ, and tolR were generated and their ability to support TonB function determined. When only ExbB/D was present, activity mirrored that of the wild-type strain, but was diminished when only TolQ/R was present. Low activity was evident for the mixed complex ExbB/TolR, but not for the TolQ/ExbD complex. In vivo cross-linking indicated that ExbB interacted with TonB independent of the presence of either ExbD or TolR. TonB stability is greatly decreased in the absence of the ExbB/D complex. Here, ExbB alone, or with TolR, was sufficient for the stability of TonB. TolQ alone, or with ExbD, also stabilized TonB, despite the absence of ExbB. Together, these data suggest that the ExbB/TolQ component of a given complex is sufficient for interaction with TonB, but the ability to couple TonB to the ion gradient of the membrane requires interactions between the ExbB/TolQ and ExbD/TolR components of the complex. These data suggest that instability results from activity and TonB interactions at the CM are dependent upon varying conformational states. Like TonB, TolA is most efficient when it interacts with its heterologous energy harvesting complex (TolQ/R) and in its absence is less efficiently supported by ExbB/D. Because TonB and TolA each have a “preferred” energy-harvesting complex, it is clear motifs not shared between TonB and TolA are involved in interactions with energy harvesting complexes. Testing two distinct TolA/TonB chimeric proteins provides a different view of how the transducers in the TolA and TonB systems interact with the energy harvesting complexes. These new data suggest that the transmembrane domains of these two transducers may not be solely responsible for energization.
Brinkman, Kerry, "USING CHIMERAS TO EVALUATE CROSS-TALK, ENERGY TRANSFER, AND PROTEIN-PROTEIN INTERACTIONS IN THE TONB AND TOLA SYSTEMS" (2007). Biology Ph.D. Dissertations. 12.