Date of Award

Spring 5-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type



EmrE is a small multidrug resistance transporter in E. coli. It effluxes a wide range of antibiotics, thus contributing to the evolving epidemic of drug resistance. Despite its small size, EmrE is a fully functional transporter making it an ideal model system for a comprehensive study of the multidrug transport mechanism. In the transport cycle, EmrE must alternate between outward- and inward-facing conformations upon substrate binding to translocate substrates across the membrane. High-resolution structures of EmrE in complex with substrates facing different sides of the membrane will shed light on the coupling mechanism between substrate binding and transport. However, the conformational plasticity that enables EmrE to transport diverse drugs also makes it a very challenging system for high-resolution structural studies. The conformational dynamics inherent in the transport process require experimental measures of structural transitions to provide the link between static structures and functional transport. This thesis aims to characterize the structure and dynamics of EmrE in atomic detail using NMR, a well-established technique to study structure and dynamics of biomolecules simultaneously under a variety of conditions.

In the case of EmrE, NMR spectroscopy is the best approach for high-resolution structures because the dynamic nature and small size of EmrE hamper X-ray crystallography and cryoEM approaches. I have made significant progress towards a better structure of EmrE using a slow-dynamics mutant and have achieved a near complete backbone and side chain ILV methyl assignment for this highly challenging helical membrane protein system. I have also collected a large data set of distance and orientational restraints. I have also used NMR and functional assays to characterize a series of mutants located near the transmembrane helix 3 (TM3) kink and have demonstrated the important role of TM3 kink formation for the global conformational interconversion required for alternating-access. My NMR data also suggest that hydration within the transport pore may be an important property fine-tuning the rates of conformational interconversion. My NMR pH titrations show that the slow-dynamics mutant also has elevated pKa values for E14, the critical residue for proton-coupling in EmrE. This provides the first experimental evidence of the physicochemical link between proton and substrate binding and alternating-access necessary for achieving coupled transport. By correlating high-resolution structural and dynamic data with functional transport assays, this thesis provides key insights into the multidrug transport mechanism of EmrE. The principles learned for EmrE set the stage for understanding even more challenging transporters.


English (en)

Chair and Committee

Katherine A. Henzler-Wildman

Committee Members

Gaya A. Amarasinghe, Alexander B. Barnes, Kathleen B. Hall, Peng Yuan,


Permanent URL: https://doi.org/10.7936/K73R0R53