Folding in Place: Design of β-Strap Motifs to Stabilize the Folding of Hairpins with Long Loops.

Despite their pivotal role in defining antibody affinity and protein function, β-hairpins harboring long noncanonical loops remain synthetically challenging because of the large entropic penalty associated with their conformational folding. Little is known about the contribution and impact of stabilizing motifs on the folding of β-hairpins with loops of variable length and plasticity. Here, we report a design of minimalist β-straps (strap = strand + cap) that offset the entropic cost of long-loop folding. The judicious positioning of noncovalent interactions (hydrophobic cluster and salt-bridge) within the novel 8-mer β-strap design RW(V/H)W···WVWE stabilizes hairpins with up to 10-residue loops of varying degrees of plasticity (Tm up to 52 °C; 88 ± 1% folded at 18 °C). This "hyper" thermostable β-strap outperforms the previous gold-standard technology of β-strand-β-cap (16-mer) and provides a foundation for producing new classes of long hairpins as a viable and practical alternative to macrocyclic peptides.