Synthesis of the transmembrane domain of the spike protein from SARS‐CoV‐2 using solid phase peptide synthesis and determination of its oligomerization state

As of today, nearly six hundred thousand lives have been lost to the disease COVID-19 in the United States alone. This highly contagious disease has a proposed R0 value of 5.7, meaning that for every person infected, they transmit the virus to about 5 other people. This has caused researchers to expeditiously study Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Due to the state of the current pandemic, the distribution of effective vaccination and antiviral medication is paramount to global health. The Spike (S) protein in SARS-CoV-2 is largely responsible for the reproduction of this virus. The S protein consists of a receptor binding domain (RBD), which the virus uses to bind to angiotensin-converting enzyme 2 (ACE2) receptors of host cells (REF) as well as a stem like structure binding to the membrane. The fusion of the viral and cell membranes allow for the release of the viral genome. It is known that the Spike protein has a trimerized oligomerization state, which is believed to be necessary to its function. The goal of this project is then to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the S protein in SARS-CoV-2. The native sequence of this peptide is the following: WYIWLGFIAGLIAIVMVTIML In order to determine the oligomerization state of the TM Domain of the S protein, that being how many times the peptide may repeat or come together with identical units of itself. By conducting a fluorescence-based oligomerization assay this may be determined. This method relies upon having the native sequence of the domain separate from the entire protein to determine the specific effect of oligomerization state on the TM domain. The most practical method to synthesize the TM domain of the S protein is through Solid Phase Peptide Synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will additionally need to be purified and quantified through high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography-mass spectrometry (LCMS) to determine the prevalence and mass of the synthesized peptides. If the masses correlate to the same masses found in the S protein of SARS-CoV-2, then the correct peptides will have been synthesized correctly. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-211-13. The function of the S protein is believed to only present when the peptides from trimers. Therefore, the analysis of their oligomerization states, that is how well trimerization can occur, will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and TAMRA in the fluorescence assay.