Alphonse Sébastien, Itin Boris, Khayat Reza, Ghose Ranajeet
Department of Chemistry and Biochemistry , The City College of New York , 160 Convent Avenue , New York , New York 10031 , United States.
New York Structural Biology Center , 89 Convent Avenue , New York , New York 10027 , United States.
Biochemistry. 2018 Mar 13;57(10):1568-1571. doi: 10.1021/acs.biochem.8b00003. Epub 2018 Feb 23.
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 vitro capsid 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.
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质或单一多聚蛋白形成的衣壳。在多蛋白病毒衣壳中选择性标记单个蛋白质成分的能力以及由此产生的光谱简化将有助于将ssNMR技术扩展到复杂病毒。通过将单独纯化、标记和未标记的成分以适合核磁共振研究的量组合进行体外衣壳组装，对于大多数病毒来说不是一个可行的选择。为了克服这一障碍，我们提出了一种方法，该方法利用顺序蛋白表达和在细胞内组装成分特异性标记的病毒衣壳，其数量适合核磁共振研究。我们应用这种方法纯化仅在其四种组成蛋白成分之一——NTPase P4上进行同位素标记的噬菌体ϕ6的衣壳。使用P4标记的ϕ6衣壳以及动态核极化提供的灵敏度增强，我们展示了该方法在实现复杂病毒的ssNMR研究方面的效用。
