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Title page for ETD etd-04192010-162108


Type of Document Dissertation
Author Amadi, Sepan Tariq Hassan
URN etd-04192010-162108
Title Structure, dynamics and substrate-induced conformational changes of Escherichia coli multidrug transporter (EmrE)
Degree PhD
Department Molecular Physiology and Biophysics
Advisory Committee
Advisor Name Title
Alber Beth Committee Chair
Borden Lacy Committee Member
Charles Cobb Committee Member
Phoebe Stewart Committee Member
Tony Weil Committee Member
Keywords
  • EPR
  • TRANSPORTER
  • MULTIDRUG RESISTANCE
Date of Defense 2010-02-26
Availability unrestricted
Abstract
The multidrug transporter EmrE is a proton coupled secondary transporter from Escherichia coli. EmrE confers resistance to a variety of positively charged hydrophobic cations by removing them out of the cell in exchange for protons. The mechanism of substrate transport has been well characterized. The conformational changes that initiate the movement of the substrate across the membrane have not been examined. The goal of this work was to investigate the structure and dynamics of EmrE using electron paramagnetic resonance (EPR) spectroscopy.

The data set consists of the mobilities, solvent accessibilities of each spin labeled sites along the full length of the protein. These parameters were interpreted as constraints on the local steric environment, the orientation of helices in the lipid phase and packing of the transmembrane (TM) helices in the dimer. Dipolar coupling across the dimer interface of TM1, TM2 and TM3 reveal multiple spin label populations, suggesting different packing of EmrE monomers. Binding of tetraphenyl phosphonium (TPP+) to the protein reduces dipolar coupling between symmetry related spin label pairs located in the middle of TM1. In contrast, for sites located in the middle of TM2 and the N-terminal region of TM3 report increased dipolar coupling. In parallel, the N-terminus region of TM2 is slightly tilted and move away from the dimer interface. These changes are in support with the alternating access model. Moreover, upon substrate binding an increase in the structural order is observed in the C-terminus region of TM3 and loop connecting it to TM4. The EPR analysis suggests that in liposomes the structure of these regions deviate from the antiparallel static crystal structure in detergent micelles. A model to resolve these discrepancies is proposed.

We also investigated the helix packing in EmrE monomer using double electron-electron resonance (DEER) to measure distance between two spin label sites in solution. The result reports the packing and orientation of TM2 and TM3 with respect to TM1 deviates from the crystal structure. Furthermore, multimodal distance distributions were observed for symmetry related sites specifically in TM3. The overall consensus of the distance measurements confirms the dynamic flexibility of EmrE backbone in the apo-state and decreases upon substrate binding.

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