| Abstract
| - Amino acid structural propensities measured in “host−guest” model studies are often used inprotein structure prediction or to choose appropriate residues in de novo protein design. While this concepthas proven useful for helical structures, it is more difficult to apply successfully to β-sheets. We havedeveloped a cyclic β-hairpin scaffold as a host for measurement of individual residue contributions to hairpinstructural stability. Previously, we have characterized substitutions in non-backbone-hydrogen-bonded strandsites; relative stability differences measured in the cyclic host are highly predictive of changes in foldingfree energy for linear β-hairpin peptides. Here, we examine the hydrogen-bonded strand positions of ourhost. Surprisingly, we find a large favorable contribution to stability from a valine (or isoleucine) substitutionimmediately preceding the C-terminal cysteine of the host peptide, but not at the cross-strand position ofthe host or in either strand of a folded linear β-hairpin (trpzip peptide). Further substitutions in the peptidesand NMR structural analysis indicate that the stabilizing effect of valine is general for CX8C cyclic hairpinsand cannot be explained by particular side-chain−side-chain interactions. Instead, a localized decrease intwist of the peptide backbone on the N-terminal side of the cysteine allows the valine side chain to adopta unique conformation that decreases the solvent accessibility of the peptide backbone. The conformationdiffers from the highly twisted (coiled) conformation of the trpzip hairpins and is more typical of conformationspresent in multistranded β-sheets. This unexpected structural fine-tuning may explain why cyclic hairpinsselected from phage-displayed libraries often have valine in the same position, preceding the C-terminalcysteine. It also emphasizes the diversity of structures accessible to β-strands and the importance ofconsidering not only “β-propensity”, but also hydrogen-bonding pattern and strand twist, when designing βstructures. Finally, we observe correlated, cooperative stabilization from side-chain substitutions on oppositefaces of the hairpin. This suggests that cooperative folding in β-hairpins and other small β-structures isdriven by cooperative strand−strand association.
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