NMR analysis of tuning cross-strand Phe/Tyr/Trp–Trp interactions in designed β-hairpin peptides: terminal switch from l to D amino acid as a strategy for β-hairpin capping

Abstract

Interaction among the side chains of aromatic amino acids is a well-known mechanism of protein and peptide structure stabilization, particularly in β sheets. Using short β-hairpin models bearing the sequence Ac-Leu-Xxx-Val-DPro-Gly-Leu-Trp-Val-NH₂, we report the surprising observation of significant destabilization in aryl–tryptophan interactions, which results in poorly folded peptide populations accompanied by lowering of stability. We find that such destabilization arises from forced occupancy of the indole ring in the shielded Edge position, in T-shaped aryl geometries. We demonstrate that this destabilizing effect can be efficiently salvaged by replacing the N-terminal LLeu with DLeu, which causes an increase in the folded hairpin population, while retaining Trp in the Edge position. Our observation of unique cross strand NOEs and data from temperature-dependent NMR and CD measurements reveals the formation of a locally stabilized aliphatic–aromatic network, leading to an overall increase in ΔG_F° by ∼ −0.6 to −1.2 kcal/mol. Our results suggest that a contextual evaluation of stabilization by tryptophan is necessary in β hairpins. Furthermore, we report for the first time that the use of D isomers of aliphatic amino acids at the terminus is stabilizing, which can serve as a new strategy for increasing β-hairpin stability.