Designing and stabilising peptide-protein interactions

Christensen, Pernille Vosbein (2024) Designing and stabilising peptide-protein interactions. PhD thesis, University of Glasgow.

Abstract

Protein-protein interactions are important for both disease treatment and understanding of biological processes. These interactions are often targeted using peptides which mimic larger protein domains. This thesis describes the development of α-helical coiled coil mimics.

A two heptad repeat scaffold coiled coil was made using triazole ligation. The most stable coiled coil was obtained when linking the peptides through an a-position with respect to the coiled coil register. This coiled coil scaffold was used to target two biologically relevant PPIs: The p53:MDM2 interaction and the Vps27 UIM-1:ubiquitin interaction. Designed MDM2-binding coiled coils showed improved structural stability and similar binding affinity when compared to their parent peptides.

The placement of a fluorescent label was found to greatly affect the binding affinity of the UIM-1 peptide to ubiquitin. An N-terminal label significantly increased the binding energy. Experimental alanine scanning of the UIM-1 peptide revealed that the most important hydrophobic residues for the binding interaction are UIM-1 residues Ile7 and Ile11, and that residues Leu6 and Leu13 contribute the least to the overall binding energy. A ‘decoy’ AlphaFold competition assay was able to replicate the ranking of the hydrophobic residues obtained in the experimental alanine scan. This AlphaFold assay was thus able to discriminate between single point mutation binders with small differences in binding energy.

It was possible to add binding residues from UIM-1 onto the scaffold coiled coil, designing a novel ubiquitin-binding coiled coil with improved binding affinity compared to the native UIM-1 peptide. A crystal structure of the ubiquitin-binding coiled coil showed that this peptide successfully mimicked the native UIM-1: ubiquitin interaction.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Thomson, Dr. Andrew R.
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84342
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 05 Jun 2024 10:35
• Last Modified: 05 Jun 2024 10:38
• Thesis DOI: 10.5525/gla.thesis.84342
• URI: https://theses.gla.ac.uk/id/eprint/84342