Early Events in Protein Folding

蛋白质折叠的早期事件

• 批准号: 9027085
• 负责人: RICHARD BRIAN DYER
• 金额: $ 14.99万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9027085
• 关键词: Acids; Affect; Antiviral Agents; Biochemical; Biological Models; Cells; Collaborations; Complex; Coupled; Development; Equilibrium; Event; Extravasation; Fluorescence; Fluorescence Spectroscopy; Grant; Hemagglutinin; Higher Order Chromatin Structure; Infection prevention; Influenza; Influenza Hemagglutinin; Laboratories; Lasers; Lead; Lipid Bilayers; Lipids; Mediating; Membrane; Membrane Fluidity; Membrane Fusion; Membrane Lipids; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Molecular Models; Mutation; N-terminal; Nature; Peptides; Process; Proteins; Reaction; Reaction Time; Research; Series; Specificity; Structure; Temperature; Testing; Time; Vesicle; Viral; Virus; Virus Diseases; Work; base; drug development; improved; interest; molecular dynamics; molecular modeling; novel strategies; protein folding; public health relevance; research study; scaffold; simulation; temperature jump; time use

DESCRIPTION (provided by applicant): Enveloped viruses infect target host cells through protein mediated membrane fusion. Influenza hemagglutinin (HA) has served as the paradigm for understanding the general mechanism of membrane fusion and it is also an important target for antiviral drug development. The central hypothesis of the proposal is that pH induced refolding of hemagglutinin drives the fusion of the viral and host membranes. The mechanism of membrane fusion has been inferred from equilibrium structures of fragments of hemagglutinin, along with biochemical evidence. Hemagglutinin is postulated to undergo an astounding series of refolding reactions triggered by lowered pH, first to form an extended coiled coil conformation that exposes the fusion peptides and then an even more dramatic refolding to an antiparallel coiled-coil, six-helix bundle scaffold. Finally, a zipping of linker domains against this scaffold s postulated to drive the membranes together and facilitate membrane fusion. We plan to elucidate the molecular details of this dynamic refolding process, and the coupled interactions with lipid bilayers that accomplish membrane fusion, using time-resolved spectroscopic methods that we have developed to study protein folding in membranes. Our approach will focus on all of the critical components of the complex mechanism, including the pH induced formation of an extended coiled-coil by the trigger peptide region (L40) and the insertion of the fusion peptide into the host membrane to start the fusion process. We will study these two functional units in the soluble HA2 domain to determine how these processes are coupled. We will also determine if the complete soluble hemagglutinin protein refolds to form an antiparallel coiled-coil structure coupled to these earlier steps, followed by the refolding of the linker domains against this scaffold. These studies will make use of laser induced pH and temperature jump methods pioneered in our laboratory, as well as ultrafast mixing to rapidly initiate the HA refolding reaction, and time resolved IR and fluorescence spectroscopy to follow the dynamics with high structural specificity. We expect that our unique approach combined with our focus on HA as an archetype will provide an unprecedented molecular view of the dynamic function of this important class of protein machines, and thereby improve our understanding of protein mediated membrane fusion. More generally, we expect a better understanding of the dynamics and molecular mechanisms of protein folding in membranes to emerge from this work. Protein folding at the boundary of or within the membrane is a process that has been very difficult to study and as a consequence is poorly understood. As a basis for understanding the dynamic interactions of HA with the lipid bilayer, we propose to study fundamental folding processes of model systems at the interface of or within membranes, including membrane association, insertion, folding, and assembly into higher order structures.

描述（由申请人提供）：包裹的病毒通过蛋白质介导的膜融合感染靶宿主细胞。流感血凝素（HA）已成为理解膜融合的一般机制的范式，它也是抗病毒药物发育的重要靶标。该提案的中心假设是，pH诱导的血凝素蛋白重折叠驱动病毒和宿主膜的融合。膜融合的机理是从血凝素片段的平衡结构以及生化证据中推断出来的。假定血凝素会经历一系列令人震惊的重折叠反应，该反应是由降低的pH触发的，首先形成延伸的盘绕线圈构象，暴露于融合肽，然后将更具戏剧性的重新折叠到反平行的盘旋围墙，六螺旋束支架上。最后，接头域的拉链针对该脚手架的S，以驱动膜并促进膜融合。我们计划使用时间分辨的光谱方法来阐明这种动态重折叠过程的分子细节，并与脂质双层相互作用，并使用时间分辨的光谱方法进行了研究，我们已经开发了我们开发的来研究膜中蛋白质折叠的方法。我们的方法将集中于复杂机制的所有关键组成部分，包括触发肽区域（L40）的pH诱导形成延伸的盘绕螺旋，以及将融合肽插入到宿主膜中以开始融合过程。我们将研究可溶性HA2域中的这两个功能单元，以确定这些过程的耦合方式。我们还将确定是否会重新折叠完整的可溶性血凝素蛋白以形成反平行线圈结构 结合了这些早期步骤，然后将接头域的重新折叠针对此脚手架。这些研究将利用激光诱导的pH和温度跳跃方法，并在我们的实验室中率先使用，以及超快混合以迅速引发HA重折叠反应，时间解决了IR和荧光光谱，以遵循具有高结构特异性的动力学。我们期望我们的独特方法与对HA的关注相结合，将其作为原型，将为这种重要的蛋白质机器的动态功能提供前所未有的分子视图，从而提高了我们对蛋白质介导的膜融合的理解。更普遍地，我们期望更好地了解膜中蛋白质折叠的动力学和分子机制，从而从这项工作中出现。膜的边界或膜内的蛋白质折叠是一个很难研究的过程，因此对众所周知。作为理解HA与脂质双层的动态相互作用的基础，我们建议研究膜或膜内或内部模型系统的基本折叠过程，包括膜关联，插入，折叠和组装到高阶结构中。