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The Molecular Mechanism of the Escherichia Coli vitamin B12 Transporter BtuCD-F: Real-time Observation of the Transporter in Motion

Kim, Jinrang

The Escherichia coli vitamin B12 transporter BtuCD-F is a type II importer belonging to the ABC transporter superfamily. Available data suggest both exporters and type I importers in the ABC superfamily employ similar transport mechanism in which the transmembrane (TMDs) are open to cytoplasm in the resting state, and ATP binding induces a major conformational change resulting in opening of the TMDs instead to the periplasm. However, the crystal structures of BtuCD from E. coli and recent EPR spectroscopy studies indicate that this type II importer employs a substantially different mechanism in which the TMDs are open to the periplasm in the resting state and to the cytoplasm after ATP binding. We have developed robust methods to study the conformation and transport mechanism of BtuCD-F reconstituted into lipid bilayers using single molecule fluorescence resonance energy transfer (smFRET) measurements. Fluorescent probes have been introduced at a variety of diagnostic sites, enabling smFRET to be used to measure distance changes in different conformational states as well as to observe the transitions between these states in real time. These data suggest that thermal fluctuations enable the transporter to explore different functional conformational states in the absence of ATP or other ligands. They also suggest that the ATP-bound state is indeed open to the cytoplasm and ATP binding/hydrolysis increases the rate of transition between open and closed states. Efforts are currently underway to observe the transport of vitamin B12 through a single BtuCD-F oligomer in real-time.

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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Hunt, John F.
Degree
Ph.D., Columbia University
Published Here
August 27, 2012
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