Interplay between hydrophobic cluster and loop propensity in β-hairpin formation

Autonomously folding β-hairpins have recently emerged as powerful tools for elucidating the origins of antiparallel β-sheet folding preferences. Analysis of such model systems has suggested four potential sources of β-sheet stability: (1) the conformational propensity of the loop segment that connects adjacent strands; (2) favorable contacts between side-chains on adjacent strands; (3) interstrand hydrogen bonds; and (4) the intrinsic β-sheet propensities of the strand residues. We describe the design and analysis of a series of isomeric 20 residue peptides in which factors (1)-(4) are identical. Differences in β-hairpin formation within this series demonstrate that these four factors, individually, are not sufficient to explain β-sheet stability. In agreement with the prediction of a simple statistical mechanical model for β-hairpin formation, our results show that the separation between the loop segment and an interstrand cluster of hydrophobic side-chains strongly influences β-hairpin size and stability, with a smaller separation leading to greater stability.