Abstract

Macrocyclization, or stapling, has emerged as a powerful strategy to enhance the stability and function of peptides and proteins by constraining their conformational ensembles. Stapling can improve thermodynamic stability, increase resistance to proteolytic degradation, and enhance biomolecular recognition, making it particularly valuable for the development of peptide-based therapeutics. Despite these advantages, staple design remains largely empirical, relying on iterative screening of linker chemistries, lengths, and attachment sites. This trial-and-error approach limits scalability and hinders the rational design of stabilized peptide systems. In this work, we address this challenge by integrating large-scale experimental datasets with molecular dynamic simulations to develop predictive guidelines for staple design. We demonstrate that staple-induced stabilization can be understood through a balance of two key effects: unfolded-state constraint and folded-state compatibility. Unfolded-state constraint describes the entropic advantage gained when a staple restricts the spatial separation of residues in the unfolded ensemble, while folded-state compatibility reflects how well the staple's maximum span aligns with the native residue separation in the folded structure. Together, these principles provide a unifying framework for rational staple placement and linker selection prior to experimental validation. Chapter 1 introduces the principles of peptide stapling, with an emphasis on applications beyond traditional coiled-coil systems, and reviews current design strategies across diverse structural scaffolds. Chapter 2 systematically evaluates staple placement within coiled-coil peptides and establishes design guidelines governing optimal attachment sites. Chapter 3 investigates the role of linker length in modulating stability, extending these analyses from coiled-coil systems to a helix-bundle HER2 affibody model to assess generalizability. Chapter 4 presents the design, synthesis, and preliminary characterization of stapled HER2 affibody variants, including binding affinity measurements that evaluate functional consequences of stapling. Finally, Chapter 5 describes a collaborative study with the Andrew Roberts group at the University of Utah, incorporating a novel staple chemistry into coiled-coil systems to further probe the relationship between linker properties and peptide stability. Collectively, this work establishes a predictive framework for peptide stapling that integrates experimental and computational approaches, enabling more efficient and rational design of stabilized peptide architectures.

Degree

PhD

College and Department

Computational, Mathematical, and Physical Sciences; Chemistry and Biochemistry

Rights

https://lib.byu.edu/about/copyright/