Development of In vitro Quantitative Förster Resonance Energy Transfer (qFRET) Reporter Assays for the Characterization of Non-Covalent Interaction of Membrane Proteins and Covalent SUMOylation Modification of Viral Proteins Coupled with Mass Spectrometry Identification of SUMO1 Modified Lysine
Disease progression is often facilitated by protein-protein interaction. Reports of commandeering host protein mechanisms by cancer and viral proteins has armed scientist with novel drug targets. The challenge arises when attempting to quantitatively evaluate the variety of covalent and non-covalent interactions that occur during disease progression. This thesis covers the development of a quantitative Förster resonance energy transfer (qFRET) assay for the evaluation of covalent SUMOylation of viral proteins and non-covalent interaction of programmed cell death 1 (PD1) with programmed cell death ligand 1 (PDL1). The interaction of PD1 and PDL1 results in the negative regulation of T cell immune response and is reported to be used by cancer cells to circumvent the immune checkpoint mechanism. Immune checkpoints inhibitors, that target PD1-PDL1 interaction, have been the focus for cancer therapies in the past decade, but recent development of novel treatments has plateaued. Reported here is a FRET based reporter for the in vitro characterization of the PD1 and PDL1 interaction. The results of the work produced a qFRET based KD of full-length custom codon optimized PD1 and PDL1 of 0.82 µM with standard error of 0.13 µM and determined a qFRET based Ki of PermbrolizumabTM to be 1.14 nM with a 95 % confidence interval of 0.94 to 2.14 nM. The remainder of the work focuses on the development of an in vitro SUMOylation of viral proteins with qFRET as a reporter, in combination with mass spectrometry to identify SUMO1 modified lysine sites. The qFRET assay for the in vitro SUMOylation of IAV-Matrix Protein 1 in tandem with MS, resolved 5 SUMO1 modification. The evaluation of the novel K21 modification proved fatal to the viral pathogenesis, and M1 K242R mutant reported diminished IAV infectivity. The qFRET based in vitro SUMOylation of severe acute respiratory syndrome coronavirus 2 Nucleocapsid protein (SARS-CoV- 2 N) coupled with MS analysis identified 4 novel SUMO modified lysine residues. The evaluation of identified lysine in vitro and in cell mutant studies found lysine 61 and 65 to modulate SUMOylation activity and modulate cellular translocation.