Using Hydrogen Bond Surrogate Technology to Stabilize Beta-Hairpins

Abstract

Beta-hairpins are comprised of two β-strands connected by a short hairpin loop. The β-hairpin motif is critical in many protein folds and diverse protein-protein interactions (PPIs). Extensive studies on the forces governing β-hairpin folding and stability have led to the development of many strategies for β-hairpin stabilization, including side-chain cross-linking, rigid loop mimics, and macrocyclization. While these strategies have provided valuable insight into β-hairpin behavior, sacrifice of side chains may limit their application in the design and evaluation of β-hairpin PPI inhibitors. To overcome these challenges, we investigated the hydrogen bond surrogate (HBS) strategy as a method for β-hairpin stabilization. Previous studies have demonstrated the effectiveness of the HBS strategy in nucleation and stabilization of peptides in the α-helical conformation. Alpha-helix nucleation is accomplished by replacing the N-terminal backbone hydrogen bond with a covalent bond using ring-closing olefin metathesis on alkene substrates positioned at the i and i+4 peptide positions. Importantly, mimicry of a backbone hydrogen bond yields peptides with a full complement of side-chain functional groups. Our model system is a well-characterized β-hairpin derived from the B1 domain of protein G (GB1). The HBS macrocycle was formed on solid support by joining alkenes that mimic a hydrogen bond between the peptide's N- and C-termini. Conformational stabilities of unconstrained and HBS peptides were evaluated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Consistent with previous results, we find that the unconstrained peptides show weak propensity for the β-hairpin conformation while HBS macrocyclization significantly increases β-hairpin propensity. We envision that the HBS strategy will be generally effective in stabilizing diverse β-hairpin sequences.