Title: Two-Way Transmembrane Signaling in an Outer-Membrane Transport Protein: or why do spectroscopy when we have a crystal structure?
Abstract: Proteins execute motion over a wide range of amplitudes and time scales, which may include large amplitude movements between discrete structural substates. A change in the equilibrium distribution of these substates is thought to underlie protein allostery and to play a role in mediating protein-protein recognition. Site-directed spin labeling when combined with EPR spectroscopy is a powerful method to examine conformational exchange and structural heterogeneity in globular, membrane proteins and protein complexes. We will discuss TonB-dependent transporters, which are an important class of outer-membrane transport proteins that function in the uptake of nutrients by Gram negative bacteria. In particular, we will describe the use of EPR spectroscopy to characterize the Escherichia coli vitamin B12 transporter, BtuB, and show that the Ton box (an N-terminal energy coupling motif) and the substrate binding site are allosterically coupled. We will discuss the likely mechanisms of transport in this family of transport proteins, and describe novel methods to achieve efficient spin-labeling and carry out pulse EPR spectroscopy on these proteins in whole bacteria.