Advancing our knowledge of viral membrane fusion and of IDP-membrane interactions by ESR

多年来，弗里德一直是研究电子自旋共振(ESR)方法的先驱 蛋白质及其对细胞膜的动力学效应。这项提议的目标是使用这些ESR技术 增进我们对1)病毒膜融合和2)内源性无序蛋白(IDP)膜的认识 与其他生物物理方法相结合的相互作用。 正在进行的COVID19大流行揭示了我们抗击的知识和方法是多么有限 由病毒病原体引起的新发传染病。SARS-CoV-2(SARS-2)病毒感染的一个关键步骤是 膜融合是由其Spike蛋白中的融合多肽(FP)结构域启动的。事实上，膜融合是 所有包膜病毒，如SARS-CoV-1(“SARS-1”)、MERS-CoV、EBOV、流感和艾滋病毒的检索表。然而， 病毒膜融合的机制尚不清楚。我们已经广泛地证明了FP诱导的 在所有这些病毒中，膜有序是病毒膜融合的先决条件。我们已经证明了 包括SARS-1、MERS和EBOV在内的一些病毒的膜有序是强烈依赖于钙的。多数 最近，我们发现SARS-2FP与膜的相互作用比SARS-1更强，这取决于 非常明确地说是关于钙离子。然而，钙的确切作用以及膜有序化的机制是 仍不清楚。FP只启动膜融合。我们提出了跨膜区(TMD) 流感血凝素对于膜融合的最终确定是重要的。然而，这还有待检验。 看看它是否适用于SARS-2。因此，我们计划继续研究膜融合的机制。 SARS-2以及其他依赖钙离子的病毒。 国内流离失所者在溶液中缺乏稳定的三级结构。在膜结合后，它们可能会经历一种紊乱- 在没有稳定结构的情况下，向有序转变或仍保持不变。病毒FP就是这样的例子。这个身份证明文件- 膜相互作用将IDPs定位于其靶膜，促进与其他膜的相互作用 蛋白质，并帮助重塑细胞膜的特性。了解结构/功能关系 IDP与膜的相互作用是一个巨大的挑战，因为它们高度可变和动态 大自然。我们一直在使用复合素，一种与神经退行性变相关的关键胞吐调节因子，作为一种模型 描绘了IdP-膜结合模式。我们已经发现，来自不同生物体的复合素具有非常 不同的模式，这可能反映了它们不同的生物学功能。我们计划继续研究 几种络合物的膜结合C-末端结构域的确定机制 管理络合蛋白-膜结合模式。这些发现将对人类疗法有用。 ESR是研究SARS FFP和其他国内流离失所者动态和结构特性的强大方法，如 我们已经证明了。我们将使用我们成熟的ESR方法来实现这些目标。这将是 辅以其他生物物理方法，包括等温滴定量热法和圆二色谱。

For many years Freed has pioneered the development of electron-spin resonance (ESR) methods for the study of proteins and their dynamical effects on membranes. The goal of this proposal is to use those ESR techniques to advance our knowledge of 1) viral membrane fusion and 2) Intrinsic Disordered Protein (IDP)-membrane interactions in conjunction with other biophysical methods. The ongoing COVID19 pandemic has unveiled how limited are our knowledge and methods to combat emerging infectious diseases caused by viral pathogens. A key step in SARS-CoV-2 (“SARS-2”) viral infection is membrane fusion initiated by its fusion peptide (FP) domain in its Spike protein. In fact, membrane fusion is key for all enveloped viruses such as SARS-CoV-1 (“SARS-1”), MERS-CoV, EBOV, influenza and HIV. However, the mechanism of viral membrane fusion is still unclear. We have extensively demonstrated that FP-induced membrane ordering is a prerequisite for viral membrane fusion in all these viruses. We have shown that membrane ordering for some viruses including SARS-1, MERS and EBOV is strongly Ca2+-dependent. Most recently we have shown that SARS-2 FP interacts with membranes more strongly than SARS-1, and it depends very specifically on Ca2+. However, the exact role of Ca2+, as well as the mechanism of membrane ordering are still unclear. The FP only initiates membrane fusion. We have proposed that the transmembrane domain (TMD) of influenza hemagglutinin is important for finalizing membrane fusion. However, this remains to be tested to see if it is applicable to SARS-2. Thus, we plan to continue studies of the mechanism of membrane fusion of SARS-2 as well as other Ca2+-dependent viruses. IDPs lack a stable tertiary structure in solution. After membrane binding, they can either undergo a disorder- to-order transition or still remain in the absence of a stable structure. The viral FPs are such examples. This IDP- membrane interaction localizes IDPs to their target membranes, facilitating interactions with other membrane proteins, and helping to remodel membrane properties. Understanding the structure/function relationships underlying IDP-membrane interactions is a significant challenge because of their highly variable and dynamic nature. We have been using complexin, a key exocytosis regulator related to neurodegeneration, as a model to delineate the IDP-membrane binding mode. We have found that complexins from different organisms have very different modes, which is likely to reflect their different biological functions. We plan to continue to study the membrane-binding C-terminal domain of complexins of several species to determine the mechanisms that govern complexin-membrane binding modes. These findings will be useful for human therapies. ESR is a powerful methodology to study the dynamic and structural properties of SARS FPs and other IDPs, as we have shown. We will employ our well-developed ESR methods to achieve these goals. This will be supplemented by other biophysical methods, including Isothermal Titration Calorimetry and Circular Dichroism.