Cafiso Lab Group University of Virginia

Membrane Transport

In Escherichia coli, nutrients such as iron, vitamin B12, nickel and carbohydrates are accumulated by specific, high-affinity active transport proteins that are localized to the outer-membrane (OM). These transporters are termed “TonB-dependent,” because transport requires energy that is obtained from the inner-membrane protein TonB. Gram negative bacteria may express over 60 TonB-dependent transporters (TBDTs), which are critical to the functioning of the human microbiome as well as the success of many pathogens. Approximately 50 high-resolution structures have been obtained for over 12 different TBDTs. Nonetheless, the molecular mechanisms by which these proteins function remain poorly characterized. The lack of a well-defined mechanism for TBDT is due to the fact that intermediate structural states and conformational equilibria that underlie transport have not been revealed. Moreover, transport in these systems has never been reconstituted and structures have not been characterized in a native environment where the inner-membrane complex that energizes transport is present.

We have developed approaches to label and perform continuous wave and pulse EPR on TBDTs expressed in whole cells and intact outer membranes. These approaches have been optimized and yield excellent EPR spectra and double electron-electron resonance (DEER) data, which can be used for structural studies on the intact system under conditions where active transport is taking place. We are presently obtaining structural data that is yielding new information on the structure and intermediate states of TBDTs as well as data on the organization of these OMP in the bacterial membrane. Work on these systems is currently supported by a grant from the Institute of General Medical Sciences at the NIH.