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

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