Determination of the Oligomerization States of the Transmembrane Domains from SARS-CoV-2 Membrane Proteins

SARS-CoV-2 is the third highly infectious, highly deadly coronavirus to make the jump to humans in the last two decades.SARS-CoV-2, like other coronaviruses, is an enveloped virus with a small number of integral membrane proteins embedded in a lipid membrane.Each of these proteins has a transmembrane (TM) domain, a portion of the protein approximately 20 amino acids in length.Several proteins, including the Spike protein, are functional proteins only when trimeric-that is, three copies of the protein must self-assemble in order to carry out their function.Six proteins present in SARS-CoV-2 are known or hypothesized to have TM domains.These TM domains are highly conserved, and they are an underexplored region for potential drug discovery.To examine the oligomerization state, three versions of each peptide of several of the TM proteins have been synthesized.One has a nitrobenzodiazole (NBD) fluorophore active in membranes, one has tetramethylrhodamine (TAMRA), a quencher of NBD, and one is unlabeled.These peptides are dissolved in a detergent above its CMC, which presents the peptides in micelles previously shown to be accurate mimics of membranes for the analysis of oligomerization states of TM domains.The three variants are used in a fluorescencebased assay which monitors the change in fluorescence as a function of added quencher peptide.The shape of the resulting fluorescence curve describes the oligomerization state.We describe the results of this experiment for two TM proteins, ORF 7a and ORF 3a.