Lambert O, Boisset N, Penczek P, Lamy J, Taveau J C, Frank J, Lamy J N
Laboratoire des Protéines Complexes Université François Rabelais, Tours, France.
J Mol Biol. 1994 Apr 22;238(1):75-87. doi: 10.1006/jmbi.1994.1269.
A frozen-hydrated sample of Octopus vulgaris hemocyanin was imaged at 0 degree and 40 degrees tilt angle under low dose conditions by transmission electron microscopy. A three-dimensional reconstruction by the method of random conical tilt series produced a three-dimensional volume to which a D5 symmetry was applied. Examination of serial sections in the volume and surface representation at various thresholds allowed the five arches containing functional unit Ovg to be localized at the interdimeric subunit groove. In another set of experiments specific polyclonal antibodies were used to label functional units Ovb and Ove in the cylinder wall. The observation of the negatively stained immunocomplexes showed that Ovb is located in the external tiers of functional units and Ove in the internal tier. These results suggest that the direction of the polypeptide chains in the cylinder wall may be only partially antiparallel. A model of the quaternary structure is proposed with the following features: (1) the external tiers of functional units comprise four units each (Ova-d) coming from a single polypeptide chain; (2) the internal tier comprises two functional units from each polypeptide chain (Ove-f); (3) the interdimeric subunit arches connect the two copies of a single functional unit (Ovg) located in each polypeptide chain.
通过透射电子显微镜在低剂量条件下，对普通章鱼血蓝蛋白的冷冻水合样本在0度和40度倾斜角度下进行成像。采用随机锥形倾斜系列方法进行三维重建，生成了一个应用了D5对称性的三维体积。对该体积中的连续切片以及不同阈值下的表面表示进行检查，使得包含功能单元Ovg的五个拱能够定位在二聚体间亚基凹槽处。在另一组实验中，使用特异性多克隆抗体标记圆柱壁中的功能单元Ovb和Ove。对负染免疫复合物的观察表明，Ovb位于功能单元的外层，而Ove位于内层。这些结果表明，圆柱壁中多肽链的方向可能只是部分反平行。提出了一种具有以下特征的四级结构模型：(1)功能单元的外层各包含来自一条多肽链的四个单元（Ova-d）；(2)内层各包含来自每条多肽链的两个功能单元（Ove-f）；(3)二聚体间亚基拱连接每条多肽链中位于其中的单个功能单元（Ovg）的两个副本。
