NMR Study on the Binding of Neuropeptide Achatin-I to Phospholipid Bilayer: The Equilibrium, Location, and Peptide Conformation

Abstract

Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance 13C and high-resolution (of 0.01 Hz order) 1H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal N H 3 + group in achatin-I; the de-ionization of N H 3 + decreases the bound fraction of the peptide from ∼15% to nearly none. The electrostatic attraction between the N-terminal positive N H 3 + group and the negative P O 4 − group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the 13C chemical shifts ( δ) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Δ δ of the 13C resonance for almost all the carbon sites in achatin-I. The changes Δ δ for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic C O 2 − groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic D-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.