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

项目主任/首席调查员(Freed，Jack，H.)： 项目摘要/摘要 这是为米拉赠款(R35GM148272)的设备提供资金的补充申请，每笔资金 机会号PA-20-272。我们35卢比赠款的主要目标之一是了解结构 融合多肽(FP)诱导病毒膜融合过程的机制(S) SARS-CoV-2，埃博拉病毒等被膜病毒糖蛋白的跨膜域(TMD)， 流感病毒和艾滋病毒。我们先进的ESR技术揭示了相互作用的主要结构细节 在膜中的多肽和脂类之间，在多肽和膜上。然而，它将是信息量大的 如果我们能够同时进行功能研究来补充我们的结构研究。事实上，使用ESR，我们有 检测FP在改变膜结构中的功能，这是 膜融合过程。然而，我们需要一种方法来检测膜融合的最后一步，在这种方法中， 两个小泡的膜融合在一起。透视检查是治疗这种疾病的理想和成熟的技术。 任务。此外，该技术也可应用于我们的新型假病毒颗粒-囊泡对接系统。 我们的另一个主要目标是研究固有无序蛋白(IDP)与膜的相互作用。许多这样的人 相互作用依赖于钙，包括病毒FP。因此，钙离子-IdP结合常数的测量 不同钙离子浓度下的IDP膜参与常数是重要的，因此我们可以了解到 在我们的结构研究中，有多大比例的IDPs处于膜结合状态。透视检查是一种 获取这些参数的简便技术。通过透视获得的结果也可以进行比较。 补充和补充了我们ESR实验的平行测量结果。 OMB编号0925-0001/0002(批准的第03/2020版至2023年2月28日)续页格式页

Program Director/Principal Investigator (Freed, Jack, H.): Project Summary/Abstract This is a supplementary request for funding for equipment for the MIRA grant (R35GM148272), per Funding Opportunity PA-20-272. One of the major objectives of our R35 grant is to understand the structural mechanism(s) underlying the viral membrane fusion process induced by the fusion peptide (FP) and the transmembrane domain (TMD) of glycoproteins of an enveloped virus such as SARS-CoV-2, Ebola Virus, influenza virus, and HIV. Our advanced ESR technology has revealed major structural details of the interactions between peptides and lipids in membranes, on both the peptides and membranes. However, it will be informative if we can carry out functional studies in parallel to complement our structural studies. In fact, using ESR we have detected the function of the FP in altering the structure of the membrane, which is the initial step of the membrane fusion process. However, we need a method to detect the final step of membrane fusion, in which the membranes of the two vesicles fuse together. Fluoroscopy is the ideal and well-established technique for this task. In addition, this technique can also be applied to our novel pseudo-viral particle-vesicle docking system. Our other major objective is to study Intrinsic Disordered Protein (IDP)-membrane interactions. Many such interactions are calcium dependent, including the viral FP. Thus, measurements of Ca2+-IDP binding constant and the IDP-membrane participation constant at different Ca2+ concentrations are important, so we can learn what percentage of the IDPs in our structural study is in the membrane binding condition. Fluoroscopy is a convenient technique to obtain these parameters. The results obtained from fluoroscopy can also be compared to and complement those of the parallel measurements from our ESR experiments. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page