Cerritelli M E, Studier F W
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
J Mol Biol. 1996 May 3;258(2):286-98. doi: 10.1006/jmbi.1996.0250.
Prohead-like capsid shells containing the scaffolding and head proteins of bacteriophage T7 were isolated after both proteins were expressed from the cloned genes in the same cell. When the head-tail connector protein was also expressed, the isolated capsids contained neither connector nor scaffolding protein and resembled mature phage capsids rather than proheads. However, only a small fraction of the head protein was converted to stable capsid structures in either case. Purified scaffolding protein (expressed individually from the cloned gene) appeared to be a monomer in solution; purified head protein appeared to be a tetramer. The purified proteins reacted in the presence of polyethylene glycol or dextran to produce prohead-like capsid shells and also polycapsids consisting primarily of head protein, similar to the polycapsids observed after infection by T7 mutants lacking connector or core proteins. Neither capsids nor polycapsids were produced in the absence of scaffolding protein. Polycapsids were usually the predominant product even when scaffolding protein was in excess, and a small fraction of scaffolding protein catalyzed the conversion of an excess of head protein to polycapsids. Our results suggest that the first step in the natural pathway to prohead formation is the assembly of incomplete prohead shells, which are normally closed by insertion of a connector-core complex. In the absence of a functional connector-core complex, incomplete capsid shells apparently react further to form polycapsids or completely closed capsid shells.
在噬菌体T7的支架蛋白和头部蛋白在同一细胞中从克隆基因表达后，分离得到了含有这两种蛋白的类原头部衣壳壳。当头部-尾部连接蛋白也表达时，分离得到的衣壳既不含有连接蛋白也不含有支架蛋白，并且类似于成熟的噬菌体衣壳而非原头部。然而，在这两种情况下，只有一小部分头部蛋白转化为稳定的衣壳结构。纯化的支架蛋白（从克隆基因单独表达）在溶液中似乎是单体；纯化的头部蛋白似乎是四聚体。纯化的蛋白在聚乙二醇或葡聚糖存在下反应，产生类原头部衣壳壳以及主要由头部蛋白组成的多衣壳，类似于在缺乏连接蛋白或核心蛋白的T7突变体感染后观察到的多衣壳。在没有支架蛋白的情况下，既不产生衣壳也不产生多衣壳。即使支架蛋白过量，多衣壳通常也是主要产物，并且一小部分支架蛋白催化过量的头部蛋白转化为多衣壳。我们的结果表明，原头部形成自然途径的第一步是不完全原头部壳的组装，这些壳通常通过插入连接蛋白-核心复合物而封闭。在没有功能性连接蛋白-核心复合物的情况下，不完全衣壳壳显然会进一步反应形成多衣壳或完全封闭的衣壳壳。
