White Dawn, Musse Abdiwahab A, Wang Jie, London Erwin, Merrill A Rod
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
J Biol Chem. 2006 Oct 27;281(43):32375-84. doi: 10.1074/jbc.M605880200. Epub 2006 Jul 19.
The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic alpha-helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic alpha-helix (3.8 +/- 0.1 residues per turn and 94 +/- 4 degrees, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr(363)-Gly(364)) and that short amphipathic alpha-helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.
通过定点荧光标记法，利用附着在每个突变蛋白螺旋2内每个半胱氨酸残基上的双硫腙荧光团，研究了大肠杆菌素E1通道结构域的膜结合封闭状态。测量了封闭通道膜结合形式下双硫腙荧光团的荧光特性，包括最大荧光发射、荧光各向异性、表观极性、表面可及性和膜双层穿透深度。荧光数据表明，螺旋2是一个两亲性α螺旋，与膜表面平行，但在封闭通道中，它比螺旋1嵌入双层界面区域的深度要浅。将各种数据集与谐波函数进行最小二乘拟合表明，膜结合状态下螺旋2的周期性和角频率是位于膜双层界面区域的两亲性α螺旋的典型特征（分别为每圈3.8±0.1个残基和94±4度）。双淬灭剂分析还表明，螺旋2与膜外周相关，螺旋的一面浸入脂质双层的界面区域，另一面则向外伸入水相溶剂中。最后，我们的数据表明，螺旋1和螺旋2在与短连接链（Tyr(363)-Gly(364)）膜结合时仍保持独立螺旋，并且短两亲性α螺旋参与了这种大肠杆菌素的脂质依赖性环形孔的形成。
