Taylor R M, Zakharov S D, Bernard Heymann J, Girvin M E, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Biochemistry. 2000 Oct 10;39(40):12131-9. doi: 10.1021/bi000206c.
The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions.
大肠杆菌素E1免疫蛋白（ImmE1）是一种位于大肠杆菌细胞质膜中的13.2 kDa疏水整合膜蛋白，可保护细胞免受细菌素大肠杆菌素E1的致死性通道形成活性的影响。利用其在有机溶剂中的溶解性，通过用1-丁醇提取分离的膜来纯化ImmE1，随后在氯仿/甲醇/H₂O（4:4:1）溶剂系统中进行凝胶过滤和离子交换色谱。圆二色性分析表明，ImmE1在1-丁醇或2,2,2-三氟乙醇中的α-螺旋含量约为80%，这与先前具有三个延伸疏水跨膜螺旋结构域H1-H3的膜折叠模型一致。这些延伸疏水结构域中的每一个都包含一个位于中心的单个半胱氨酸残基，可作为蛋白质结构的探针。纯化的ImmE1在混合极性溶剂氯仿/甲醇/H₂O（4:4:1）中的三级结构的存在通过以下方式得到证明：（i）在270-285 nm波长区间近紫外圆二色性光谱振幅所显示的对酪氨酸残基的限制；（ii）Imm蛋白的单半胱氨酸和双半胱氨酸到X突变体的近紫外酪氨酸CD光谱与其体内活性之间的相关性；（iii）在混合极性溶剂混合物中ImmE1的一维NMR光谱中甲基的上移、二维HSQC NMR光谱，以及通过连接到H2疏水结构域中Cys16的自旋标记使H3中Trp94的吲哚（1）H质子共振变宽和消失；（iv）从插入延伸疏水结构域的单个色氨酸得到的以290 nm为中心的具有突出椭圆率带的近紫外圆二色性光谱。得出的结论是，大肠杆菌素E1免疫蛋白在氯仿/甲醇/H₂O（4:4:1）中采用通过螺旋-螺旋相互作用稳定的折叠构象。对可能的膜折叠拓扑结构的分析表明，三个跨膜螺旋双层区域中的几个酪氨酸残基可通过螺旋-螺旋相互作用对近紫外CD光谱有贡献。
