Abstract 2193: Synthesis of the Transmembrane Domain of the Spike Protein from SARS-CoV-2 Using Solid Phase Peptide Synthesis and Determination of Its Oligomerization State

Abstract


Abstract 2190 pH Effects on the Stability and Folding of Monomeric Caspases
Isha Joglekar, University of Texas at Arlington

Clay Clark
Caspases are an ancient class of cysteinyl proteases that are integral in cell development and apoptosis as an evolutionarily conserved function. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies: initiators, which are monomeric, and effectors, which are dimeric. Regulation of apoptosis is influenced by the activation mechanism of these two subfamilies, and how the subfamilies of monomers versus the dimers evolved from a well-conserved caspase-hemoglobinase fold is not well understood. We examined the folding landscape of monomeric caspases from two coral species, 300 million years distant on an evolutionary timescale between themselves and about 600 million years distant from human caspases. Since these coral caspases do not unfold via a distinct mechanism at all pH studied, the pH dependence on equilibrium unfolding is complex. Our results indicate that both proteins have overall high stability ∼ 16 kcal mol-1 near the physiological pH range ( pH 6 to pH 8) and unfold through a four-state mechanism via two intermediate states. The intermediate state (I2) is not sensitive to changes in pH, and the folding mechanism is mainly dependent on the stabilization of the native state and the intermediate (I1) state. Outside of this pH range, the native state is destabilized, whereas the intermediate (I1) is destabilized at extreme pH. Hence, the four-state model shifts to a three-state and a twostate as an effect of pH. We observe a decrease in the average emission wavelength of the native proteins and a decrease in the overall conformational stability below pH 6, which can be characterized by an estimated pKa ∼ 5.7. This suggests a pHdependent conformational change due to the protonation of a histidine residue, also observed in the dimeric family of caspases. Together, the data suggest that the folding landscape is conserved, with a possibly conserved allosteric mechanism. Furthermore, urea MD simulations data paired with limited proteolysis and MALDI-TOF indicate that the small subunit of monomeric caspases is unstable and could be the first to unfold, suggesting the importance of the evolution of the subfamily of stable dimers.
I would like to thank NIH-NIGMS for funding this research.

Timothy Reichart
The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as a whole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. 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 then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography-mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. 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-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay.
-Hampden-Sydney College Office of Undergraduate Research.