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Examination of EmrE conformational differences in various membrane mimetic environments
Sandra L Federkeil1, Tara L Winstone, Glen Jickling
1Division of Biochemistry, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1N4, Canada.
Summary
Investigating the Ethidium multidrug resistance protein (EmrE) in various membrane mimetics reveals its tryptophan residues are most flexible in methanol. Detergent environments, particularly N-dodecyl-beta-D-maltoside (DM), best mimic lipid environments for EmrE studies.
Area of Science:
- Biochemistry
- Structural Biology
- Membrane Proteins
Background:
- Ethidium multidrug resistance protein (EmrE) confers resistance in Escherichia coli to lipophilic cations.
- Understanding EmrE's structure and function is crucial for combating multidrug resistance.
- The choice of membrane mimetic significantly impacts studies on membrane protein structure and dynamics.
Purpose of the Study:
- To investigate the influence of different membrane mimetic environments on the structure and dynamics of EmrE.
- To compare EmrE's properties in various solubilizing conditions using fluorescence spectroscopy and circular dichroism.
- To determine the optimal environment for future structural, folding, and ligand binding studies of EmrE.
Main Methods:
- Solubilization of EmrE in diverse membrane mimetic environments including methanol, sodium dodecyl sulfate, urea, N-dodecyl-beta-D-maltoside (DM), polyoxyethylene(8)dodecyl ether, trifluoroethanol, and small unilamellar vesicles.
- Fluorescence spectroscopy to analyze emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization.
- Circular dichroism (CD) spectroscopy to monitor secondary structure, specifically alpha-helical content.
Main Results:
- Tryptophan residues of EmrE exhibit the highest flexibility and exposure in methanol, followed by sodium dodecyl sulfate and urea.
- N-dodecyl-beta-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether show subtle spectral differences, with DM best representing the lipid environment.
- EmrE adopts a more open and dynamic conformation in detergent solutions compared to reconstituted small unilamellar vesicles.
- Trifluoroethanol provides an environment similar to detergents for EmrE.
- Circular dichroism spectra indicate consistent alpha-helical content across different solubilizing conditions.
Conclusions:
- The choice of membrane mimetic significantly influences the conformational state and dynamics of EmrE.
- Detergent environments, especially DM, are suitable for studying EmrE's lipid-like interactions.
- These findings provide essential groundwork for selecting appropriate conditions for future structural and functional investigations of EmrE.