Link, A. JamesJung, Seyi2024-07-232026-09-292024-07-232026-09-292024-05-142024-07-23http://arks.princeton.edu/ark:/88435/dsp012z10wt59mhttps://theses-dissertations.princeton.edu/handle/88435/dsp012z10wt59mGraspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing side chain-side chain ester/amide linkages. Fuscimiditide is a specific graspetide with a modifiable loop region connected to a stem with two ester linkages. The ester-linked stem stabilizes the peptide within the loop region and is protease resistant, making the fuscimiditide an ideal candidate for epitope grafting. This project focused on grafting P29S1, a peptide sequence that inhibits the SARS-CoV-2 Spike RBD interaction with the ACE2 receptor1, onto the fuscimiditide stem. Five different designs were constructed in total, and the locations of their ester linkages were determined by tandem mass spectrometry (MS/MS). The final design, fusP29S1_D15N_R13A, with T3V and D24N substitutions in the fuscimiditide stem along with R13A and D15N in the P29S1 epitope, resulted in an ester linkage between Thr7 of the stem and Asp16 of the epitope. Even though this ester linkage was undesired, the peptide’s binding affinity (KD) against the SARS-CoV-2 Spike RBD was still measured using biolayer interferometry (BLI). The BLI assay of the peptide shows no effective binding to the Spike RBD relative to the positive control, the antibody casirivimab. Even so, this paper presents the challenges in grafting an epitope onto fuscimiditide and offers various improvement strategies for future designs.application/pdfenPrinceton University Senior Theses