Human opioid peptide Met-enkephalin binds to anionic phosphatidylserine in high preference to zwitterionic phosphatidylcholine: natural-abundance 13C NMR study on the binding state in large unilamellar vesicles

A human opioid neuropeptide, Met-enkephalin (M-Enk: Tyr1-Gly2-Gly3-Phe4-Met5), having no net charge binds to anionic phosphatidylserine (PS) in high preference to zwitterionic phosphatidylcholine (PC). The binding mechanism in the PS and PC bilayers was studied on the basis of the inter- and intramolecular interaction data obtained by natural-abundance 13C nuclear magnetic resonance (NMR) of the peptide. Prominent upfield changes of the 13C resonance were observed in the C-terminal residue upon binding to PS, whereas no such marked change was observed upon binding to PC. The upfield chemical shift changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide C-terminal CO2- and the PS headgroup NH3+. Despite the net negative charge of the PS bilayer surface, M-Enk thus anchors the negatively charged C-terminus. In the N-terminal residue, on the other hand, marked downfield chemical shift changes are observed upon binding to both the PS and PC bilayers, the magnitude of the changes being much larger in the PS system. The downfield changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup negative charge(s) (CO2- or PO4- in PS, PO4- in PC). Perturbation on the signal half-widths due to membrane binding also indicates the preferential and deeper binding of M-Enk on the PS membrane surface than on the PC membrane surface. Local charge cancellation takes place efficiently between M-Enk termini and the PS headgroups and compensates for the strong electrostatic hydration of the ionic groups. Distribution of the charged (positive and negative) and uncharged sites in the headgroups along the bilayer normal is responsible for the marked difference between PS and PC headgroups in controlling the binding state of the zwitterionic M-Enk.