AbstractSynthesis of HIV-1 GP41 Including FP and MPER by Native Chemical Ligation with Applications to SSNMR. Expression, Solubilization, and Purification of SARS-CoV-2 Spike Protein Subunit 2 By Rob Wolfe Human immunodeficiency virus type 1 (HIV-1) and Coronavirus 2019 (Covid-19) have caused substantial risk to public health worldwide. Both HIV-1 and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are membrane enveloped viruses which cause acquired immune deficiency syndrome (AIDS) and COVID-19 in humans, respectively. The process by which these viruses initiate the infection by membrane fusion of the viral and host cells is similar. However, they differ in which membrane protein is responsible for the fusion process. The HIV-1 glycoprotein 41 (Gp41) is a single-pass integral viral membrane protein containing a ~170-residue ectodomain that is important for membrane fusion between virus and host cells. The ectodomain includes the fusion peptide (FP), N-helical region (NHR), loop, C-helical region (CHR) and viral membrane-proximal external region (MPER). The ectodomain mediates joining (fusion) of the HIV-1 and host cell membranes, which is in an initial step in infection. The ectodomain also adopts different structures, including a final hyper thermostable state. Some data supports a fusion role for this final state. Like HIV-1, SARS2-CoV-2 is enveloped by a membrane that is obtained during budding from an infected host cell. Infection of a new cell requires fusion of the virus membrane with a membrane of the target cell and subsequent deposition of the viral nucleocapsid in the cytoplasm. This process is catalyzed by the Spike (S) protein subunit 2 (S2). My research has focused on the production and characterization of several protein constructs (with or without FP) and the characterization of S2_816-1273, a large S2 protein construct containing all regions including the FP, transmembrane (TM) and the cytoplasmic tail (CT). The expression vector used is pET-24a(+). Biophysical characterization comparisons between S2_816-1273 and shorter constructs including S2_903-998SGGRGG1153-1207 and S2_903-998SGGRGG1163-1207 were performed. Biophysical characterization techniques such as circular dichroism (CD) spectroscopy, size exclusion chromatography (SEC), mass spectrometry (MS), and vesicle fusion assays were used in this study. In addition, this work also describes development and application of native chemical ligation (NCL) methods to produce most of the HIV glycoprotein-41 (gp41) ectodomain. NCL with yields up to 45% were achieved between the chemically synthesized N-terminal FP (gp41512-534) and bacterially synthesized hairpin with MPER (HM) (gp41535-581/SGGRGG/628-683) segments. The hairpin is the NHR-loop-CHR region. These high yields were achieved by solubilizing the HM segment in guanidinium chloride and imidazole which worked together to fully denature the reactants, facilitating the NCL reaction via exposure of the reactant termini. HM was produced with fractional 13C or 2H-labelings that were respectively in the 14-82% and 18% to 54% ranges. The synthesized FP included a non-native N-terminal H6G6D4K tag, which allowed for the binding of FP-HM to a metal-affinity column at the Nterminus to facilitate the separation of unreacted HM. FP-HM was then released from the column by enterokinase-catalyzed cleavage. My work will help future scientists synthesize site-specific 13C and 2H labeled large protein constructs since this is the first time that large Gp41 constructs including full MPER have been synthesized in mg quantities using a combination of bacterial expression system and solid phase peptide synthesis. Furthermore, this methodology is applicable to many proteins that cannot be easily characterized by other methods inside of the lipid bilayer, such as crystallography.
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