Early Events in Protein Folding

描述（由申请人提供）：包膜病毒通过蛋白介导的膜融合感染靶宿主细胞。流感血凝素（flu hemagglutinin， HA）已成为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。该提议的中心假设是pH诱导血凝素的再折叠驱动病毒和宿主膜的融合。膜融合的机制已经从血凝素片段的平衡结构以及生化证据中推断出来。据推测，血凝素在降低pH值的情况下会发生一系列令人震惊的再折叠反应，首先形成一个延长的盘绕状结构，暴露融合肽，然后更戏剧性的再折叠成一个反平行的盘绕状结构，六螺旋束支架。最后，连接体结构域在支架上的压缩被假设为驱动膜在一起并促进膜融合。我们计划阐明这种动态重折叠过程的分子细节，以及与脂质双分子层的耦合相互作用，完成膜融合，使用我们开发的时间分辨光谱方法来研究膜中的蛋白质折叠。我们的方法将专注于复杂机制的所有关键组成部分，包括pH诱导的由触发肽区（L40）形成的延伸卷曲线圈，以及融合肽插入宿主膜以开始融合过程。我们将研究可溶性HA2结构域中的这两个功能单元，以确定这些过程是如何耦合的。我们还将确定是否完全可溶性血凝素蛋白重新折叠，形成一个反平行卷曲的线圈结构

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.