Molecular order, dynamics, and ionization state of phosphatidylethanolamine bilayers as studied by nitrogen-15 NMR

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Abstract
Dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylphosphatidylcholine (DPPC), 15N-labeled in the polar head group, were synthesized. The proton-decoupled 15N spectra of DPPC and DPPE in aqueous dispersion have exactly the form anticipated for powder line shapes governed by an axially symmetric shielding tensor. The chemical shift anisotropy (.DELTA..sigma.) of DPPC is lower than 0.4 ppm at 30 .degree.C and vanished when the temperature or the half-height line width is increased; DPPE always exhibits an asymmetric line shape, and 15N NMR spectra of DPPE are obtained at various temperatures and simulated to measure exactly the chemical shift anisotropy. At each temperature, the order parameter of the C.sbd.N bond segment is derivated from .DELTA..sigma. and reveals that the average orientation of the C.sbd.N bond around the axis of rotation is near the "magic angle" (54.7.degree.). Isotropic correlation times are derived from T1, which are higher than values obtained for phosphatidylcholine by other nuclei. Arrhenius plots of T1 and T2 allowed us to calculate the activation energy for the motion of the DPPE and the DPPC C.sbd.N bond. The value of this activation energy for the DPPE (53 kJ/mol) is higher than the one found for the DPPC C .sbd. N bond (32 kJ/mol). These differences agree with the capacity of the ethanolamine head groups to bind noncovalently to their neighbors in the plane of the membrane surface. A direct titration curve of the amino group is achieved by the variation of the chemical shift with the bulk pH, and the interfacial pKa is calculated to be 11.1. At pH 11, two distinct protonation states of DPPE are observed, which are in slow exchange compared to the NMR time scale. The present results clearly show the great discriminating power of 15N spectroscopy in terms of environmental changes around the nitrogen atom at the interfacial region of membranes.