Federkeil Sandra L, Winstone Tara L, Jickling Glen, Turner Raymond J
Division of Biochemistry, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB T2N 1N4, Canada.
Biochem Cell Biol. 2003 Apr;81(2):61-70. doi: 10.1139/o03-031.
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.
溴化乙锭多药耐药蛋白（EmrE）是小多药耐药蛋白家族的成员之一，负责大肠杆菌对多种亲脂性阳离子的耐药性。研究开始阐明该蛋白的结构信息以及底物结合和转运机制。然而，需要选择合适的膜模拟环境来进行结构研究。在本研究中，将EmrE溶解在不同的膜模拟环境中，通过比较发射最大值、峰位移、相对强度、丙烯酰胺猝灭常数和极化的荧光特性，研究环境对该蛋白结构和动力学的影响。综合来看，在测试的各种膜模拟系统中对EmrE的不同荧光观察结果表明，当溶解在甲醇中时，EmrE中的色氨酸残基处于最灵活和暴露的状态，其次是十二烷基硫酸钠和尿素。两种去污剂N-十二烷基-β-D-麦芽糖苷（DM）和聚氧乙烯（8）十二烷基醚，在光谱特性上大多只表现出细微差异，其中DM最能代表脂质环境。相对于重构在小单层囊泡中，EmrE在去污剂中的构象明显更开放和动态。溶解在三氟乙醇中的EmrE的荧光观察结果显示其环境与溶解在去污剂中的EmrE相似。此外，通过圆二色性（CD）监测二级结构。在不同的溶解条件下，CD光谱相似，表明α-螺旋含量差异不大。这项工作为未来结构、折叠和配体结合研究的溶解条件选择奠定了基础。
