Early Events in Protein Folding

描述（由申请方提供）：包膜病毒通过蛋白质介导的膜融合感染靶宿主细胞。流感病毒血凝素（HA）是了解膜融合一般机制的范例，也是抗病毒药物开发的重要靶点。该提案的中心假设是pH诱导的血凝素重折叠驱动病毒和宿主膜的融合。从血凝素片段的平衡结构，沿着生化证据，已经推断出膜融合的机制。假设血凝素经历了一系列令人震惊的重折叠反应，由降低的pH值触发，首先形成一个延长的卷曲螺旋构象，暴露融合肽，然后更戏剧性的重折叠到一个反平行卷曲螺旋，六螺旋束支架。最后，假定连接结构域对该支架的压缩将膜驱动在一起并促进膜融合。我们计划使用我们为研究膜中蛋白质折叠而开发的时间分辨光谱方法，阐明这种动态重折叠过程的分子细节，以及与实现膜融合的脂双层的偶联相互作用。我们的方法将集中在复杂机制的所有关键组成部分，包括pH诱导的触发肽区域（L40）的延伸卷曲螺旋的形成和融合肽插入宿主膜开始融合过程。我们将研究这两个功能单位在可溶性HA2域，以确定这些过程是如何耦合。我们还将确定是否完整的可溶性血凝素蛋白重折叠形成反平行卷曲螺旋结构 偶联到这些较早的步骤，接着是针对该支架的接头结构域的重折叠。这些研究将利用我们实验室开创的激光诱导pH和温度跳跃方法，以及超快混合来快速启动HA重折叠反应，以及时间分辨IR和荧光光谱来跟踪具有高结构特异性的动力学。我们预计，我们独特的方法与我们对HA作为原型的关注相结合，将为这一类重要蛋白质机器的动态功能提供前所未有的分子视角，从而提高我们对蛋白质介导的膜融合的理解。更一般地说，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，从这项工作中出现。蛋白质在膜的边界或膜内的折叠是一个非常难以研究的过程，因此对其了解甚少。作为理解HA与脂质双层的动态相互作用的基础，我们建议研究模型系统在膜的界面处或膜内的基本折叠过程，包括膜缔合、插入、折叠和组装成高阶结构。

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.