Prevelige P E, Thomas D, King J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Mol Biol. 1988 Aug 20;202(4):743-57. doi: 10.1016/0022-2836(88)90555-4.
Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.
源自P22原衣壳的衣壳亚基和支架亚基已被纯化，其形式能在天然条件下于体外快速且高效地共同组装成二十面体外壳。该反应在21摄氏度下的半衰期约为五分钟。体外反应呈现出在体内观察到的调控特征。单独的衣壳亚基或支架亚基都不会自组装成大的结构。将这些亚基混合在一起后，它们会聚合成具有体内衣壳和支架蛋白组成的原衣壳样外壳。纯化的衣壳蛋白制剂中的亚基是单体形式。支架亚基似乎是单体或二聚体形式。这些结果证实，P22原衣壳的形成不是通过先组装一个支架核心，然后由该核心组织衣壳来进行的，而是需要衣壳和支架的共聚。为了探究聚合控制的机制，研究了外壳组装作为支架亚基与衣壳亚基输入比例的函数关系。结果表明，支架蛋白对于外壳组装的起始和持续聚合都是必需的。尽管体内产生的原衣壳含有约300个支架分子，但亚基数量较少的外壳也能组装，每个外壳的支架亚基下限约为140个。这些实验的总体结果表明，衣壳亚基和支架亚基在外壳组装的起始阶段和生长阶段都必须相互作用。然而，目前尚不清楚在生长过程中，衣壳亚基和支架亚基是在添加到外壳之前形成混合寡聚体，还是在生长边缘发生这种情况。
