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.

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（EV）引起人类出血热，死亡率很高。尽管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个表面跨膜糖蛋白（GP）通过介导病毒和宿主细胞膜之间的合并和融合而在进入靶细胞中起核心作用。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GP 1和GP 2。 EV的一个特殊特征是位于GP 2结构域中的内部融合肽（IFP），其含有约16个不带电荷的疏水残基（残基524  539)这对于病毒融合肽家族是典型的。据认为，在融合活性状态下，GP 2蛋白将其内在FP插入靶细胞的膜中，这是融合过程中的关键阶段。尽管从文献中可以获得，但对EV融合机制没有完全的理解。此外，与N-末端融合肽相反，通常内部融合肽在其与膜的相互作用和可能的结构作用方面的特征很差。 鉴于上述情况，我们对具有代表天然产生的埃博拉病毒融合区的氨基酸序列GAAIGLAWIPYFGPAA的合成肽进行了体外脉冲偶极ESR研究，并对两种修饰进行了取代Ala 2Cys或Ala 2至Cys和Ala 15至Cys。半胱氨酸的突变使得有可能将顺磁性自旋标记物附着到这些残基上，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，不同的构象结合模拟膜可以确定和表征。