Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

VIRAL PROTEIN-LIPID INTERACTIONS ILLUSTRATED BY THE INFLUENZA A M2 AND HEPATITIS C VIRUS CORE PROTEINS Peter P Borbat 1 ; Griffin Sanders 2 ; Adeyemi Ogunbowale 2 ; Elka R Georgieva 2 ; 1 Cornell University, Chemistry and Chemical Biology, Ithaca, NY, USA 2 Texas Tech University, Chemistry and Biochemistry, Lubbock, TX, USA We present our results on the interactions of influenza A M2 (IM2) and the hepatitis C virus (HCV) core proteins with lipid membranes. IM2 has single transmembrane (TM) helix and assembles in a homotetramer with proton channel activity. HCV core, critical for virus assembly and budding, has two domains binding RNA and lipid, respectively. We probed the assembly of the IM2 TM domain C-terminal region (TM helix and juxtamembrane residues) reconstituted into DOPC/DOPS liposomes and separated E. coli membranes containing the native lipids and proteins (i.e. protein crowding conditions). We mutated to cysteine and spin-labeled the residue L43C located at the end of the TM helix in the polar region and studied it by continuous wave (CW) ESR and double electron-electron resonance (DEER). We obtained similar results for DOPC/DOPS and E. coli membranes at pH 7.4. The CW ESR spectra showed the label in very slow-motional regime, indicating stable and tight assembly of the TM helix bundle at the lipid to-solvent boundary. The DEER results analysis yielded the distance distributions with narrow peaks at 1.68 nm and 2.37 nm. The distance and amplitude ratios of 1.41±0.2 and 2:1 were as expected for four spin labels located at the corners of a square, indicative of an axially symmetric and rigid M2 tetramer. Furthermore, DEER was applied to samples of spin-labeled L43C IM2 in E. coli membranes, using protein-to-lipid molar ratios ranging from 1:230 to 1:10,400, to reveal that IM2 tetramer is likely to assemble via a dimer intermediate, well in line with our previous results based on different spin-labeling site. Finally, we present our data on recently produced, purified, and interacted with liposomes full-length HCV core protein. Our preliminary results from negative staining EM indicate that upon binding to liposome surface, the HCV core induces membrane deformation and possibly tubulation.


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