Exploring the Mechanisms of Outer Membrane Transport through CW-EPR and High Resolution NMR
Biophysical Society Thematic Meeting, Warszawa, Poland, September 17-21, 2014
TonB dependent transporters are a family of proteins that is essential for the success of many pathogenic bacteria, making these interesting targets for new antibiotics. TonB coupling to these proteins allows utilization of the proton-motive force of the inner membrane through a mechanism not yet fully understood. The Eschericha coli vitamin B12 transporter, BtuB, is a beta barrel outer membrane transporter and the focus of this work.
Although high-resolution crystal structures have been obtained for this protein, the mechanism of substrate transport is still unclear. These structures provide no information on the transient conformational states that play a big role in transport and crystallographical conditions result in an incorrect picture of the protein’s native state. Utilizing a combination of site-directed spin labeling and chemical denaturation we are able to characterize the energetics of the excited protein conformational states that facilitate transport. Previous studies have shown that one region of the N-terminus requires significantly less energy to unfold the goal of this work has been to define the structure of this unfolded intermediate. We focused in on the hatch domain, which occludes the barrel, with the goal of understanding the energetic landscape of this domain and nature of the B12 pore.
Additionally we use high resolution NMR to probe conformational changes directly under a variety of conditions and to understand substrate mediated motion and trapped states that might occur during transport. Taken together, these techniques provide the means to elucidate the transport mechanisms for this whole family of transporters.
Conference Proceeding Title
Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Sikora, Arthur, "Exploring the Mechanisms of Outer Membrane Transport through CW-EPR and High Resolution NMR" (2014). Chemistry and Physics Faculty Proceedings, Presentations, Speeches, Lectures. 228.