NMR Investigation of the Electrostatic Effect in Binding of a Neuropeptide, Achatin-I, to Phosphatidylcholine Bilayers

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 ¹⁵N (at the N-terminus) and natural-abundance ¹³C NMR spectroscopy. Upon binding to the PC membrane, the ¹⁵N 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 β-carboxyl group in Asp4 is deionized at acidic pH, the ¹⁵N 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^-. ¹³C chemical shifts in the hydrophobic d-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain β-CO₂^- in Asp4 is deionized; the deionization of Asp4 β-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 β-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⁺.