A Hybrid Platform to Explore Chemically Modified Peptide-Protein Interactions

• Ryan Stewart, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical & Biomolecular Engineering, University of Delaware

Genetic code expansion enables site-specific installation of noncanonical amino acids (ncAAs) with diverse chemical functionalities, creating new opportunities to engineer peptide properties and protein interactions. However, determining how these defined chemical changes alter molecular recognition, especially in a library context, remains challenging. Modification and its resulting binding activity are typically measured using separate, low-throughput assays. To address this gap, we are designing a modular hybrid bacterial-yeast surface-display platform that allows for a customizable, higher-throughput system for studying genetically encoded peptides and their interactions with eukaryotic proteins. As a proof of concept, we selected enzymatic serine phosphorylation, a well‑characterized post‑translational modification with known binding partners, to validate the system before expanding to ncAAs. We first assessed signal peptide display in E. coli  by immunolabeling an affinity tag and performing flow cytometry. Fusing the tag to the display anchor’s C-terminus resulted in a low detection signal, whereas N-terminal tagging supported robust signal peptide display and detection. Through in vitro phosphorylation and a site-specific anti-signal-peptide antibody, we corroborated our platform’s ability to express phosphorylated peptides, observing phosphorylation in 80% of the bacteria-displayed signal peptides. We then tested whether each component was functional by measuring the binding of soluble biotinylated R18 (a peptide with high binding affinity to all isoforms of a phosphoserine-binding receptor protein) to a yeast-displayed phosphoserine-binding receptor protein and assessing the recognition of phosphorylated signal peptides by a receptor protein binding motif antibody. With this information, we hope to confirm that our display platform’s components are ready for coincubation and peptide-protein binding. Upon success, future work includes probing peptides encoding  ncAAs.