Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session II
41 – POS
Board 13
Exploring the Mechanisms of Outer Membrane Transport through CW-EPR and High
Resolution NMR
Arthur Sikora
.
University of Virginia, Charlottesville, USA.
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. 1 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 unfold2 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.
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