Achatin-I (Gly1-D-Phe2-Ala3-Asp4), known as a neuropeptide containing a D-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+ (Kimura, T.; Okamura, E.; Matubayasi, N.; Asami, K.; Nakahara, M. Biophys. J. 2004, 87, 375-385). To gain mechanistic insights into how the binding equilibrium is delicately controlled by the ionization state of the N-terminal amino group, peptide-lipid binding interactions are investigated by selectively enriched N-15 (at the N-terminus) and natural-abundance C-13 NMR spectroscopy. Upon binding to the PC membrane, the N-15 NMR of the N-terminal NH3+ shifts upfield. This observation supports a mechanism that the role of the N-terminal NH3+ in stabilizing the binding state is through electrostatic attraction with a headgroup negative charge, i.e., PO4-. Interestingly, when the side chain beta-carboxyl group in Asp4 is deionized at acidic pH, the N-15 signal of the N-terminal NH3+ exhibits no significant chemical-shift change upon membrane binding of achatin-I. The Asp4 side chain thus regulates efficiency of the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4-. C-13 chemical shifts in the hydrophobic D-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain beta-CO2- in Asp4 is deionized; the deionization of Asp4 beta-CO2- increases the net hydrophobicity of achatin-I with a reduction of both the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)(3)(+) in the most superficial region of the PC membrane, resulting in deeper anchoring of the phenyl ring. Hence, the electrostatic effect of the side chain beta-CO2- in Asp4 floats achatin-I on the PC membrane surface, and the binding equilibrium is sensitively controlled by the ionization state of the N-terminal NH3+.