PULSED DIPOLAR ESR STUDY ON MEMBRANE OF EBOLA VIRUS FUSION PEPTIDE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 埃博拉病毒(Ebola Virus，EV)可引起人类出血热，病死率高。虽然大量的研究工作已经应用于了解病毒感染的机制，更具体地说，病毒融合到宿主细胞的机制，但它仍然没有被揭示。到目前为止，EV具有病毒融合的共同特征：一个单一的表面跨膜糖蛋白(GP)，属于I类病毒融合蛋白，通过介导病毒和宿主细胞膜之间的合并和融合，在进入靶细胞过程中发挥核心作用。埃博拉GP表达为单链前体，翻译后被切割成两个二硫键连接的片段GP1和GP2。 EV的一个特殊特征是位于GP2结构域的内部融合肽(IFP)，它含有大约16个不带电荷的疏水残基(残基524 539)，这在病毒融合肽家族中是典型的。目前认为，在融合活性状态下，GP2蛋白将其固有的FP插入到靶细胞的细胞膜中，这是融合过程中的关键阶段。尽管从文献中可以得到什么，但对EV融合机制还没有完全的了解。此外，与N末端融合肽相比，内部融合肽一般在与膜的相互作用和可能的结构作用方面表现不佳。 对此，我们进行了体外脉冲偶极ESR研究，合成了一个氨基酸序列为GAAIGLAWIPYFGPAA的合成肽，它代表了自然积累的埃博拉病毒融合区，以及两个用取代的Ala2Cys或Ala2修饰为Cys和Ala15to Cys的修饰。半胱氨酸的突变使顺磁性自旋标记物与这些残基结合成为可能，并通过测量这些自旋标记物之间的距离来获得结构信息。因此，可以确定和表征与模拟膜结合时的不同构象。

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 (EV) causes hemorrhagic fevers in humans with a high mortality. Although significant research effort have benapplied to understand the mechanism of viral infection, and more specifically the mechanism of viral fusion into the host cell, it remains unrevealed. It is known, to date, that EV shares the common properties of viral fusion: A single surface transmembrane glycoprotein (GP), belonging to class I viral fusion proteins plays a central role in the entry into the target cells by mediating the merging and fusion between the viral and the host cell membranes. The Ebola GP, expressed as a single-chain precursor, is cleaved posttranslational into two disulfide linked fragments GP1 and GP2. A specific feature of EV is the internal fusion peptide (IFP) located in the GP2 domain, containing approximately sixteen uncharged hydrophobic residues (residues 524  539) that is typical for a family of viral fusion peptides. It is thought that in the fusion active state the GP2 protein inserts its intrinsic FP into the membrane of the target cell and this is a critical stage in the fusion process. Despite what is available from the literature, there is no complete understanding of the EV fusion mechanism. Moreover, in contrast to N-terminal fusion peptides, generally the internal fusion peptides are poorly characterized in their interaction with membranes and possible structural role. In regard to above, we have undertaken an in vitro pulsed dipolar ESR study on a synthetic peptide with the amino acid sequence GAAIGLAWIPYFGPAA, representing the naturally accruing Ebola virus fusion region, and two modifications with substituted Ala2Cys or Ala2 to Cys and Ala15to Cys. Mutations to cysteines makes it possible to attach paramagnetic spin-labels to these residues and obtain structural information by measuring distances between these spin-labels. Hence, different conformations upon binding to mimetic membranes could be determined and characterized.