Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques.
Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques.
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细胞中的顺序蛋白质表达和衣壳组装：利用固态核磁共振技术研究多蛋白病毒衣壳。
DOI：
10.1021/acs.biochem.8b00003
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Ghose,Ranajeet
中科院分区：
文献类型：
--
作者：
Alphonse,Sébastien;Itin,Boris;Khayat,Reza;Ghose,Ranajeet
虽然固态核磁共振（ssNMR）已成为研究病毒衣壳的强大技术，但目前的研究仅限于由单一蛋白或单一多聚蛋白形成的衣壳。选择性标记多蛋白病毒衣壳内的单个蛋白组分的能力和由此产生的光谱简化将促进ssNMR技术扩展到复杂的virus.In体外衣壳组装结合单独纯化，标记，和未标记的组件在NMR量是不是一个可行的选择，大多数病毒。为了克服这一障碍，我们提出了一种方法，利用顺序蛋白质表达和细胞组装的组件特异性标记的病毒衣壳的量适合于NMR研究。我们应用这种方法来纯化噬菌体p16的衣壳同位素标记上只有一个其四个组成蛋白质组分，NTFIP 4。使用P4-标记的NH 46衣壳和动态核极化提供的灵敏度增强，我们说明了这种方法的实用性，使复杂病毒的ssNMR研究。
While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses.In vitrocapsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage ϕ6 isotopically labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled ϕ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization, we illustrate the utility of this method to enable ssNMR studies of complex viruses.