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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 埃博拉病毒（EV）引起高死亡率的人类出血性发作。尽管大量的研究工作已经努力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，但它仍然未被揭示。迄今为止，EV具有病毒融合的共同特性众所周知：属于I类病毒融合蛋白的单个表面跨膜糖蛋白（GP）通过介导病毒和宿主细胞之间的融合和融合来介导进入目标细胞中的核心作用。将埃博拉病毒GP表示为单链前体，在翻译后裂解为两个二硫键链接的片段GP1和GP2。 EV的一个特定特征是位于GP2结构域中的内部融合肽（IFP），其中包含大约16个未充电的疏水残基（残基524 539），是病毒融合肽家族的典型特征。人们认为，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。尽管文献可以从文献中获得，但对EV融合机制没有完全的了解。此外，与N末端融合肽相比，通常内部融合肽在与膜和可能的结构作用方面的相互作用表征很差。 关于上述，我们已经对具有氨基酸序列Gaaiglawipyfgpaa的合成肽进行了体外脉冲偶极性ESR研究，代表了自然造成的埃博拉病毒融合区，并用质量为ala2cys或Ala2Cys或Ala2对Cys和Ala15to Cys进行了两种修饰。对半胱氨酸的突变使得可以将顺磁性自旋标签附着在这些残基上，并通过测量这些自旋标签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。