PULSED DIPOLAR ESR STUDY ON MEMBRANE OF EBOLA VIRUS FUSION PEPTIDE

埃博拉病毒融合肽膜的脉冲偶极ESR研究

• 批准号: 8364030
• 负责人: ELKA R GEORGIEVA
• 金额: $ 3.59万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364030
• 关键词: Amino Acid Sequence; Binding; Cell membrane; Cells; Cleaved cell; Cysteine; Disulfides; Ebola virus; Family; Funding; Glycoproteins; Grant; Human; In Vitro; Link; Literature; Measures; Mediating; Membrane; Modification; Molecular Conformation; Mutation; N-terminal; National Center for Research Resources; Peptides; Physiologic pulse; Play; Principal Investigator; Process; Property; Proteins; Research; Research Infrastructure; Resources; Role; Source; Spin Labels; Staging; Surface; Technology; United States National Institutes of Health; Viral; Viral Fusion Proteins; Viral Hemorrhagic Fevers; Virus Diseases; cost; mimetics; mortality; synthetic peptide

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 埃博拉病毒（EV）会导致人类出血热，死亡率很高。尽管已经付出了大量的研究努力来了解病毒感染的机制，更具体地说是病毒融合到宿主细胞中的机制，但它仍然没有被揭示。迄今为止，已知EV具有病毒融合的共同特性：属于I类病毒融合蛋白的单一表面跨膜糖蛋白（GP）通过介导合并和融合在进入靶细胞中发挥核心作用病毒和宿主细胞膜之间。表达为单链前体的埃博拉 GP 在翻译后被切割成两个二硫键连接的片段 GP1 和 GP2。 EV 的一个具体特征是位于 GP2 结构域的内部融合肽 (IFP)，包含大约 16 个不带电荷的疏水残基（残基 524 539），这是病毒融合肽家族的典型特征。据认为，在融合活性状态下，GP2蛋白将其内在的FP插入靶细胞的膜中，这是融合过程中的关键阶段。尽管有文献记载，但人们对 EV 融合机制还没有完全了解。此外，与N端融合肽相比，内部融合肽通常在其与膜的相互作用和可能的结构作用方面的表征很差。 鉴于上述情况，我们对一种合成肽进行了体外脉冲偶极 ESR 研究，该合成肽的氨基酸序列为 GAAIGLAWIPYFGPAA，代表自然产生的埃博拉病毒融合区域，并进行了两种修饰，其中 Ala2Cys 或 Ala2 替换为 Cys，Ala15 替换为 Cys。半胱氨酸的突变使得可以将顺磁性自旋标记附着到这些残基上，并通过测量这些自旋标记之间的距离来获取结构信息。因此，可以确定和表征与模拟膜结合后的不同构象。