Energetics of Weak Interactions in a β-hairpin Peptide: Electrostatic and Hydrophobic Contributions to Stability from Lysine Salt Bridges

Analysis of the contribution of ion pairing interactions to the stability of a β-hairpin in aqueous solution has been studied quantitatively by NMR. A thermodynamic cycle has been constructed involving a combination of a single mutation (Lys→Gly) and a "pH switch" (CO2-→CO2H) to remove stepwise the contributions to stability from the interaction between the C-terminal carboxylate group of Ile16 and the side chains of Lys1 and Lys2. Turning these interactions "on" and "off" is shown to affect the chemical shifts of all residues, including those in the turn, such as to suggest that folding of the hairpin approximates to a two-state process. Two independent NMR methods have been used to analyze the thermodynamics of folding and are found to be in good agreement. Differences in hairpin stability have been analyzed in terms of an electrostatic interaction between charged groups on the terminal residues and the hydrophobic component of the Lys1 side chain: we estimate the primary electrostatic interaction to contribute 1.0−1.2 kJ mol-1 to stability, consistent with previous estimates for salt bridges in solvent-exposed sites in proteins and α-helical peptides, while the hydrophobic component is smaller but still significant (0.3−0.8 kJ mol-1). The hairpin stability is extremely sensitive to small structural perturbations (single residue mutations) or environmental changes (such as pH) providing a novel vehicle for quantitative studies of weak interactions.