Deuterium NMR to study the surface of phospholipid bilayers

Deuterons placed specifically in the head-groups of phospholipids in bilayers were studied by H-NMR, which is a non-perturbing method for directly probing the conformation and dynamics of the deuterated segments at the bilayer surface. Information about the hydrationproperties ofphosphatidvlcholine bilayers was obtained by recording the spin-lattice (T;) relaxation times of DOPC-d. and DMPC-d9 as a function of water content. Head-group mobility was demonstrated to increase with the bilayer water content up to a limiting degree of hydration beyond which the bilayer dynamics remain unchanged as the water then forms a discrete bulk phase. The titration behaviour and the dissociation constants of the primary amino group and the phosphate group ofphosphatidylethanolamine were characterized isothermally by recording the quadrupole splittings Av ofDMPE-d4 as a function of bulk pH. Each deuterated segment was found to have a highly local sensitivity towards the small changes in head-group conformation occurring in response to the protonation/deprotonation of its adjacent ionizable group. Wide-line solid state H-NMR examinations of specifically head-group deuterated lipids have provided structural and dynamic information about die surface of bilayers serving as models for biological membranes (Seelig & Seelig, 1980; Akutsu & Seelig, 1981; Sixl & Watts, 1982; Sixl & Watts, 1983; Seelig etai, 1987; Watts, 1987; Seelig etal., 1988; Watts, 1989). The labelled phospholipids are shown in Figure 1, where the arrows denote the sites of deuteration. Liquid crystalline dioleoylphosphatidylcholine (DOPC-d9) and dimyristoylphosphatidylcholine (DMPC-d9) as well an equimolar mixture of DMPC-d9 with cholesterol were used in the study of bilayer hydration. For the characterization of the ionization states of the phosphate and the amino group in dimyristoylphosphatidylethanolarnine (DMPE-d4) this lipid was mixed with equimolar DMPC to ensure the formation of extended bilayers. Experiments were performed with a homebuilt spectrometer operating at a deuterium frequency of ajlii = 55.27 MHz. The H-NMR spectrum of a liquid crystalline multilamellar lipid dispersion in water has a characteristic lineshape, a "powder pattern", to which all orientations of the C-D bond contribute with spherically weighted intensities. The quadrupole splitting (arising from the interaction of the deuterium nuclear quadrupole moment with the electric field gradient of the C-D bond) between the lineshape maxima, AvQ, is sensitive to the * || I T PpiTSPHATlPYL O-P-0-CH,-CH2-3 ETHftNntAMINE