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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04192010-162108 |
| Date | 29 June 2010 |
| Creators | Amadi, Sepan Tariq Hassan |
| Contributors | Alber Beth, Tony Weil, Phoebe Stewart, Borden Lacy, Charles Cobb |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-04192010-162108/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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