Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

MEMBRANE TECHNOLOGIES FOR STRUCTURAL DETERMINATION OF VIRUS ENTRY AND TRANSMISSION Susan K Fetics 1 ; Ariha Mehta 1 ; Salam Sammour 1 ; Jared Lindenberger 1 ; Katarzyna Janowska 1 ; Chan Soo Park 1 ; Priyamvada Acharya 1 ; 1 Duke Human Vaccine Institute, Durham, NC, USA Membrane proteins, such as receptors, located on the plasma membrane of mammalian cells, account for approximately 25 % of the human proteome yet this category of proteins represents 50 – 70 % of drug targets. Therefore, these proteins are a major focus for the development of vaccines and other therapeutics. Entry of a virus into a host cell typically involves interaction of the viral surface protein with a receptor on the host cell membrane. To facilitate structural studies of viral proteins in native-like environments, we are developing a suite of tools utilizing the full length HIV-1 Envelope (Env) protein from the JR-FL isolate as a model protein. We have expressed, purified and determined a cryo-EM structure at 6.7 Å resolution of micelle solubilized full-length HIV-1 JR-FL Env in complex with VRC01, PGT145 and DH1317.4 antibodies. However, to establish a more native membrane environment, we are developing bicelles and nanodiscs, some of which are of novel compositions. For characterization of the membrane mimetic systems alone and in the presence of Env, we use biophysical and biochemical tools, including mass photometry, differential scanning fluorimetry (DSF), dynamic light scattering (DLS), surface plasmon resonance (SPR), and electron microscopy (EM). We have expressed and purified four membrane scaffolding proteins (MSPs) of different sizes. Combined with bicelles prepared in-house, we have used these MSPs to assemble nanodiscs that mimic various cell membrane systems. Preliminary results indicate successful exchange of Env from buffer containing micelles into nanodiscs. With our current workflow, we can continue to develop membrane mimetics for applications into other human viral systems such as coronaviruses, flaviviruses, human papillomaviruses, hepatitis, and oncogenic viruses.

23