The interfacial conformation and transbilayer movement of diacylglycerols in phospholipid bilayers.

Abstract

The interaction of diacylglycerols, primarily 1,2-dilauroyl-sn-glycerol (1,2-DLG), with egg phosphatidylcholine (PC) bilayers was studied by NMR spectroscopy and other physical techniques. In the low proportions used (less than or equal to 20 mol % with respect to total lipid), 1,2-DLG formed bilayers with PC with no hexagonal phase separation, as assessed by light, polarizing and electron microscopy, and 31P and 13C NMR spectroscopy. The 13C-carbonyl chemical shift of 90% [13C]carbonyl 1,2-DLG was monitored in small unilamellar vesicles as a function of relative DLG content (1.5-20%) and temperature (10-55 degrees C). The chemically inequivalent sn-1 and sn-2 carbonyls gave a single, narrow resonance in vesicles, in contrast to neat 1,2-DLG and 1,2-DLG in organic solvents, whose spectra showed two well-separated carbonyl resonances. The chemical shift of 1,2-DLG in PC shows that the carbonyl groups are proximal to the aqueous interface, necessitating orientation of the DLG molecule along the normal to the bilayer. Both carbonyl groups are H-bonded to H2O, but the secondary ester (sn-2) carbonyl is relatively more hydrated than the primary ester (sn-1) carbonyl. The 13C-carbonyl chemical shift data further suggest that the interfacial conformation resembles that of crystalline and liquid crystalline lamellar 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine and certain PCs, in which the glycerol backbone is perpendicular to the bilayer plane. This conformation is different from that of crystalline 1,2-dilauroyl-sn-glycerol, in which the glycerol backbone is parallel to the bilayer plane. Between 1.5 and 8% DLG in vesicles, the chemical shift of the 1,2-DLG carbonyl at a given temperature was constant. However, above 8% DLG the chemical shift at each temperature increased with increasing DLG concentration, suggesting increased hydration at higher DLG content. At low temperatures 13C NMR spectra of vesicles with the highest proportions of 1,2-DLG studied (15 and 20%) showed two DLG carbonyl resonances, which most likely represent 1,2-DLG on outer and inner leaflets of the vesicle bilayer. The two peaks collapsed into a single resonance by 38 degrees C, at which temperature the two environments equilibrate with a rate constant of approximately 60 s-1 (t1/2 approximately 10 ms). Thus, transbilayer movement of DLG is extremely fast compared with phospholipids. In vesicles the 1,3-isomer of DLG exhibited a narrow carbonyl peak slightly downfield from that of 1,2-DLG. Acyl chain migration from 1,2-DLG to 1,3-DLG was monitored directly in the vesicle by time-dependent NMR measurements.