UC San Diego

The spread of novel coronavirus, SARS-CoV-2, a lethal and infectious respiratory syndrome, has heightened urgency in antiviral drug development. The SARS-CoV-2 envelope (E) and spike (S) proteins play a significant role in viral lifecycle and pathogenicity. The immediate threat posed by this infectious disease necessitates structural and functional characterization of these key structural membrane proteins involved in viral infection and replication. E protein’s transmembrane domain exhibits ion channel activity while its C-terminal domain is thought to participate in protein-protein interactions. S protein initiates viral infection through host-cell attachment and is proposed to participate in intermolecular interactions with E. Here, a bacterial expression and purification system is introduced to allow successful expression and purification of membrane proteins through E. coli. Use of this system accelerates production of purified full-length E and S proteins for spectroscopic NMR studies. Purification optimization allowed for high-resolution NMR spectra leading to characterization of the full-length E protein secondary structure and dynamics through assessment of CSPs. Interactions between E and known ion-channel inhibitor hexamethylene amiloride (HMA) allowed for characterization of the drug binding site. Analysis of the N-terminal transmembrane domain indicated Asn15 crucial to preserving E protein’s conformation. To evaluate potential intermolecular interactions between the C-terminal domains of E and S proteins, a truncated S protein construct was developed. Purification and expression of the S protein construct led to an initial 1H/15N HSQC spectrum, paving the way for future studies involving E and S protein-protein interactions integral to their structure and function in the pathogenicity of SARS-CoV-2.