STRUCTURE DETERMINATION OF EBOLA VIRUS VP35 PROTEIN BY PDS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 埃博拉病毒VP 35是一种多功能蛋白，对VP 35的毒力和人类死亡率具有重要意义。它是病毒RNA聚合酶复合体的一部分，也负责RNA沉默、抑制和RNA依赖性蛋白激酶抑制。VP 35由N端卷曲螺旋区和C端干扰素抑制结构域组成，与dsRNA结合。在过去的两年中，蛋白质的C-末端部分的两种晶体结构被释放，具有和不具有结合的8-bp dsRNA。发现该结构域在不含RNA时是单体的，或者在与dsRNA结合时结晶为二聚体的二聚体。尽管进行了几项体外和体内生物化学研究以阐明该蛋白的寡聚化状态，但没有关于全长EV VP 35的结构信息。生化方法表明存在至少四种寡聚体形式的VP 35（单体到四聚体，和三聚体/四聚体），其中一些负责EV的毒力。 我们的目标是通过脉冲偶极ESR研究全长EV VP 35的寡聚化性质，并通过对一组单突变体和双突变体的距离测量来推断其结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Ebola virus VP35 is a multifunctional protein significant to VP35 virulence and human mortality. It is a part of the viral RNA polymerase complex; also it is responsible for the RNA silencing, suppression and RNA-depended protein kinase inhibition. VP35 is composed of N-terminal coiled-coil region and C-terminal interferon inhibitory domain, which binds dsRNA. Two crystal structures of the C-terminal part of the protein were released in the last two years, with and without bound 8-bp dsRNA. It was found that this domain is monomeric when it is RNA free or else crystallizes as a dimer of dimers when bound to dsRNA. Even though several in vitro as well as in vivo biochemical studies were performed to elucidate the oligomerization states of this protein, there is no structural information on the full length EV VP35. Biochemical methods suggested the existence of at least four oligomeric forms of VP35 (monomer to tetramer, and trimer/tetramer), with some of them responsible for the virulence of the EV. Our goal is to make inquiry into the oligomerization properties of the full length EV VP35 by using pulse dipolar ESR and to follow on to infer its structure with the distance measurements on a set of single and double mutants.