The asparagine-stabilized beta-turn of apamin: Contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to expl ore the contribution of hydrogen bonds involving Asn2 to local (beta -turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retain ed a stable conformation during 2.4 ns of simulation at 67 degreesC, with h igh beta -turn stability characterized by backbone-side chain hydrogen bond s involving beta -turn residues K4 and A5, with the N2 side chain amide car bonyl. The loss of stabilizing interactions involving the N2 side chain res ulted in the loss of the beta -turn conformation in the apamin N2A simulati ons (27 or 67 degreesC). This loss of beta -turn stability propagates throu ghout the peptide structure, with destabilization of the C-terminal helix c onnected to the N-terminal region by two disulfide bonds. Backbone stabilit y in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, los s of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta -turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in ap amin N2A as compared with apamin, corresponding to free energy (delta Delta G(uf)) of around 1.5 kcal M-1 at 20 degreesC. This is equivalent to the con tribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta -turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in a pamin that account for virtually the full thermodynamic stability of the pe ptide have been quantitated.