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 蛋白质及其对膜的动力学影响。本提案的目标是使用ESR技术 为了推进我们对1）病毒膜融合和2）内在无序蛋白（IDP）-膜的认识， 与其他生物物理学方法相结合的相互作用。 持续的COVID 19大流行揭示了我们的知识和方法是多么有限，以打击 由病毒病原体引起的新发传染病。SARS-CoV-2（“SARS-2”）病毒感染的关键步骤是 由其刺突蛋白中的融合肽（FP）结构域引发的膜融合。事实上，膜融合 所有包膜病毒如SARS-CoV-1（“SARS-1”）、MERS-CoV、EBOV、流感和HIV的关键。然而，在这方面， 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 膜有序化是所有这些病毒中病毒膜融合的先决条件。我们已经证明 包括SARS-1、MERS和EBOV在内的一些病毒的膜有序化强烈依赖于Ca 2+。最 最近，我们发现SARS-2 FP与膜的相互作用比SARS-1更强，并且它依赖于 特别是Ca 2+。然而，Ca 2+的确切作用以及膜有序化的机制是未知的。 目前仍不清楚. FP仅启动膜融合。我们提出跨膜结构域（TMD） 流感血凝素的释放对于完成膜融合是重要的。然而，这仍有待检验， 看看是否适用于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Ca 2+的病毒。 国内流离失所者缺乏稳定的三级结构。在膜结合后，它们要么会发生紊乱- 向有序过渡或仍停留在缺乏稳定结构的状态。病毒FP就是这样的例子。这个IDP- 膜相互作用将IDP定位于其靶膜，促进与其他膜的相互作用， 蛋白质，并帮助重塑膜特性。理解结构/功能关系 潜在的IDP-膜相互作用是一个重大的挑战，因为它们的高度可变和动态 自然我们一直在使用复合蛋白，一种与神经变性相关的关键胞吐调节因子，作为模型， 描绘IDP-膜结合模式。我们已经发现，来自不同生物体的复合蛋白具有非常 不同的模式，这可能反映了它们不同的生物功能。我们计划继续研究 膜结合的C-末端结构域的几个物种，以确定机制， 控制复合蛋白-膜结合模式。这些发现将对人类治疗有用。 ESR是研究SARS FP和其他IDP的动力学和结构特性的有力方法， 我们已经展示了。我们将采用我们成熟的ESR方法来实现这些目标。这将是 通过其他生物物理方法，包括等温滴定量热法和圆二色性补充。

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.