Structural transitions in the scaffolding and coat proteins of P22 virus during assembly and disassembly.
Structural transitions in the scaffolding and coat proteins of P22 virus during assembly and disassembly.
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DOI：
10.1021/bi952793l
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发表时间：
1996-04
期刊：
影响因子：
2.9
通讯作者：
R. Tuma;P. Prevelige;G. Thomas
中科院分区：
文献类型：
--
作者：
R. Tuma;P. Prevelige;G. Thomas
建立了一个研究沙门氏菌噬菌体P22组装的体外系统，以阐明病毒前衣壳蛋白(Gp8)和外壳蛋白(Gp5)亚基之间识别的结构基础。用拉曼光谱和圆二色谱研究了gp8和gp5在天然前壳中的结构热稳定性，并表征了伴随着骨架退出、前壳膨胀和壳解体而发生的结构变化。研究发现，分离的GP8亚基的二级结构富含α-螺旋(约40%)，高度不耐热，具有不合作展开的特征(Tm约49℃)。相反，与proapsid结合的GP8亚单位表现出其α-螺旋二级结构的稳定性，其特征是协同展开。由于GP8的协同去折叠与Proapsid的退出相吻合，因此本结果表明去折叠和释放是耦合的过程。在监测酪氨酸和色氨酸侧链环境的拉曼标记中，不含前壳糖苷和结合前壳糖苷的GP8亚基之间的结构差异也很明显。空的proapsid壳的温度分辨拉曼光谱显示gp5亚基有三个明显的结构转变。第一种，发生在50到65摄氏度之间，归因于壳的膨胀，导致贝塔链二级结构的增加。这两次较高的温度转变分别发生在70-80℃和80-95℃之间，归因于壳亚单位的部分展开和随后的壳层解体。含有支架蛋白的proapsids也检测到相同的gp5结构转变。根据观察到的GP8打开和释放之间的热力学耦合，我们提出了一个P22 Proapsid组装的模型。讨论了该模型对dsDNA病毒体内组装的意义。
An in vitro system for investigating the assembly of the Salmonella phage P22 has been exploited to elucidate the structural basis of recognition between scaffolding protein (gp8) and coat protein (gp5) subunits of the viral procapsid. Raman spectroscopy and circular dichroism have been employed to examine structural thermostabilities of both gp8 and gp5 in native procapsids, and to characterize structural changes accompanying scaffolding exit, procapsid expansion, and shell disassembly. It is found that the secondary structure of the isolated gp8 subunit is rich in alpha-helix (approximately 40%), is highly thermolabile, and is characterized by noncooperative unfolding (Tm approximately 49 degrees C). Conversely, the procapsid-bound gp8 subunit exhibits stabilization of its alpha-helical secondary structure, characterized by cooperative unfolding. Because cooperative unfolding of gp8 coincides with exit from the procapsid, the present results suggest that unfolding and release are coupled processes. Structural differences between procapsid-free and procapsid-bound gp8 subunits are also apparent in Raman markers which monitor environments of tyrosine and tryptophan side chains. Temperature-resolved Raman spectroscopy of the empty procapsid shell reveals three distinct structural transitions for the gp5 subunits. The first, which occurs between 50 and 65 degrees C, is attributed to shell expansion and results in an increase in beta-strand secondary structure. The two higher temperature transitions, occurring within intervals of 70-80 and 80-95 degrees C, respectively, are attributed to partial unfolding of the shell subunit and subsequent shell disassembly. The same gp5 structure transitions are detected for procapsids which contain scaffolding protein. On the basis of the observed thermodynamic coupling between gp8 unfolding and its release from the procapsid, we propose a model for P22 procapsid assembly. Implications of the model for in vivo assembly of dsDNA viruses are discussed.