Early Events in Protein Folding

描述(申请人提供)：包膜病毒通过蛋白介导膜融合感染目标宿主细胞。流感血凝素(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诱导的血凝素重折叠驱动病毒和宿主膜的融合。根据血凝素片段的平衡结构，结合生化证据，推测了膜融合的机制。据推测，血凝素经历了一系列令人震惊的复性反应，由低pH引发，首先形成一个延长的卷曲构象，暴露融合多肽，然后更戏剧性的重折叠成反平行的螺旋卷曲，六螺旋束支架。最后，连接器域的拉链作用于这种支架，S认为这是为了将膜结合在一起，促进膜融合。我们计划使用我们开发的用于研究蛋白质在膜中折叠的时间分辨光谱方法，来阐明这一动态折叠过程的分子细节，以及与实现膜融合的脂质双层的耦合作用。我们的方法将集中在复杂机制的所有关键组件上，包括pH诱导触发肽区(L40)形成延伸的卷曲螺旋，以及将融合肽插入宿主膜以启动融合过程。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。我们还将确定完整的可溶性血凝素蛋白是否折叠形成反平行的卷曲结构 与这些较早的步骤相结合，随后在该支架上重新折叠连接器域。这些研究将利用我们实验室首创的激光诱导pH和温度跳跃方法，以及超快混合来快速启动HA复性反应，并利用时间分辨红外光谱和荧光光谱来跟踪具有高度结构特异性的动力学。我们期望我们独特的方法与我们对HA作为原型的关注相结合，将提供这类重要蛋白质机器动态功能的前所未有的分子观点，从而提高我们对蛋白质介导膜融合的理解。更广泛地说，我们希望通过这项工作对膜中蛋白质折叠的动力学和分子机制有更好的理解。蛋白质在膜的边界或膜内折叠是一个很难研究的过程，因此人们对此知之甚少。作为理解透明质酸与脂质双层动态相互作用的基础，我们建议研究模型体系在膜界面或膜内的基本折叠过程，包括膜的结合、插入、折叠和组装成更高阶结构。

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.