Origin of β-Hairpin Stability in Solution:  Structural and Thermodynamic Analysis of the Folding of a Model Peptide Supports Hydrophobic Stabilization in Water

The origin of the stability of isolated β-hairpins in aqueous solution is unclear with contrasting opinions as to the relative importance of interstrand hydrogen bonding, hydrophobic interactions, and conformational preferences, the latter being associated largely with the turn sequence. We have designed an unconstrained 16-residue peptide that we show folds autonomously in water to form a β-hairpin that mimics the two-stranded anti-parallel β-sheet DNA binding motif of the <i>met </i>repressor dimer. The designed peptide, with a type I‘ turn (INGK), is shown by CD and a range of NMR parameters to be appreciably folded (≈50% at 303 K) in aqueous solution with the predicted alignment of the peptide backbone. We show that the folding transition approximates to a two-state model. The hairpin has a marked temperature-dependent stability, reaching a maximum value at 303 K in water with both lower and higher temperatures destabilizing the folded structure. Van't Hoff analysis of Hα chemical shifts, reveals that folding is endothermic and entropy-driven in aqueous solution with a large negative Δ<i>C</i><sub>p</sub>, all of which are reminiscent of proteins with hydrophobic cores, pointing to the hydrophobic effect as the dominant stabilizing interaction in water. We have examined the conformational properties of the C-terminal β-strand (residues 9−16) in isolation and have shown that <sup>3</sup><i>J</i><sub>α</sub><sub>N</sub> values and backbone intra- and inter-residue Hα-NH NOE intensities deviate from those predicted for a random coil, indicating that the β-strand has a natural predisposition to adopt an extended conformation in the absence of secondary structure interactions. A family of β-hairpin structures calculated from 200 (distance and torsion angle) restraints using molecular dynamics shows that the conformation of the hairpin mimics closely the DNA binding face of the <i>met </i>repressor dimer (backbone RMSD between corresponding β-strands of 1.0 ± 0.2 Å).