title: Examination of EmrE conformational differences in various membrane mimetic environments.
abstract: Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance in Escherichia coli to a diverse group of lipophilic cations. Research is beginning to elucidate structural information as well as substrate binding and extrusion mechanisms for this protein. However, the choice of membrane mimetic environment to perform structural studies needs to be made. In this study EmrE was solubilized in different membrane mimetic environments to investigate the influence of environment on the structure and dynamics of the protein by comparing the fluorescence properties of emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization. Taken together, the different fluorescence observations on EmrE in the various membrane mimetic systems tested suggest that the tryptophan residues in EmrE are present in the most flexible and exposed state when solubilized in methanol, followed by sodium dodecyl sulfate and urea. The two detergents N-dodecyl-beta-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether, for the most part, only display subtle differences between the spectral properties with DM best representing the lipid environment. The conformation of EmrE is clearly more open and dynamic in detergent relative to being reconstituted in small unilamellar vesicles. The fluorescence observations of EmrE solubilized in trifluoroethanol shows an environment that is similar to that of EmrE solubilized in detergents. Additionally, secondary structure was monitored by circular dichroism (CD). The CD spectra were similar among the different solubilizing conditions, suggesting little difference in alpha-helical content. This work establishes groundwork for the choice of solubilizing conditions for future structural, folding, and ligand binding studies.
authors.affiliation: Division of Biochemistry, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1N4, Canada.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Federkeil
authors.forename: Tara L
authors.initials: TL
authors.lastname: Winstone
authors.forename: Glen
authors.initials: G
authors.lastname: Jickling
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Turner
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000595:Amino Acid Sequence; D017920:Antiporters / Q000138:chemical synthesis / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D002942:Circular Dichroism; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005454:Fluorescence Polarization; D006801:Humans; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C075436:EmrE protein, E coli; D020106:Acrylamide; D012967:Sodium Dodecyl Sulfate; D014364:Tryptophan