Shin I, Silman I, Weiner L M
Department of Neurobiology, Weizmann Institute of Science, Rehovol, Israel.
Protein Sci. 1996 Jan;5(1):42-51. doi: 10.1002/pro.5560050106.
A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
通过用磷脂酰肌醇特异性磷脂酶C溶解加利福尼亚电鳐电器官组织中的糖基磷脂酰肌醇锚定型乙酰胆碱酯酶，然后进行亲和层析纯化，获得了一种水溶性二聚体形式的乙酰胆碱酯酶。这种水溶性形式的酶处于催化活性的天然构象时，不与二肉豆蔻酰磷脂酰胆碱的单层囊泡相互作用。我们之前表明，化学修饰或暴露于低浓度的盐酸胍会将天然酶转化为具有熔球态物理化学特征的紧密、部分展开的形式。在本研究中，结果表明，这种熔球态形式的酶，无论是通过温和变性还是化学修饰产生的，都能与小单层囊泡有效相互作用。结合过程中没有伴随明显的囊泡融合，但在结合时会发生短暂泄漏。结合的乙酰胆碱酯酶会使囊泡的转变温度略有降低，核磁共振数据表明它主要与双层膜的头部区域相互作用。在变性条件下通过凝胶电泳监测结合的乙酰胆碱酯酶经胰蛋白酶消化后的效果。发现有一条质量约为5 kDa的单一多肽仍与囊泡结合。测序显示这是一条对应于Glu 268 - Lys 315序列的胰蛋白酶肽段。根据亲水性图谱鉴定，该多肽包含蛋白质中最长的疏水序列Leu 274 - Met 308。对乙酰胆碱酯酶三维结构的检查表明，该疏水序列在很大程度上缺乏三级结构，主要位于蛋白质表面。有人提出，该疏水序列与囊泡膜表面平行排列，非极性残基浅插入双层膜中。
