Abstract: Spike glycoprotein (S-protein) is one of the viral transmembrane proteins on the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). S- protein plays a crucial role in mediating the initial entry of viral genome into the host cell by binding to the human angiotensin-converting enzyme 2 (ACE2) and then inducing fusion between the virus envelope and cell membrane. Thus, S-protein is a target of choice for diagnostic and therapeutic assays, including neutralizing monoclonal antibodies (nAbs). To date, the conformations of S-protein and its molecular assemblies with ACE2 and/or nAbs have been mainly determined by structural techniques, including crystallographic and electron microscopic methods. These structural studies allow us to understand the molecular basis underlying viral entry and to further develop treatment and preventive therapeutics for COVID-19. However, these resolved structures are rather “static snapshots” compared to the dynamic nature of proteins in physiological conditions. Due to the technical difficulties, our knowledge about the real-time structural dynamics of S-protein and its real-time interactions with host receptors, nAbs, and the other relevant biomolecules, which may have functional significance, is still very limited. In this proposal, my lab will develop a bio-mimicking reconstitution system and apply a cutting-edge structural imaging technique, high-speed atomic force microscopy (HS-AFM), for real-time observations of S- protein’s structural dynamics in close-to-native environments and under various conditions. We will also develop novel methods to quantitatively characterize the architecture of molecular assemblies comprising S-protein, ACE2 receptor, nAbs, host proteases and enzymes, and biological membranes, which can mediate the membrane fusion and viral entry processes. Specifically, we will identify the “real-time” structural dynamics of S- protein in different states and visualize how the state transitions happen, for example, during ACE2 binding, nAbs attachment, and the structural cleavages in S-protein subunits. My lab will further develop correlated fluorescence microscopy and HS-AFM to study these dynamic events associated with S-protein on the mammalian cell surface. The biophysical and biochemical information acquired in our proposed experiments will provide a comprehensive molecular understanding of the conformational states of S-protein, intermolecular interactions between S-protein and binding molecules (ACE2 and nAbs), the conformational changes in S- protein for initiating membrane fusion processes for viral entry, and how the mammalian cell surface impacts the S-protein. The developed methods here can further apply to the other receptor-mediated membrane fusion systems for cell entry.