Nola Fuller

The Effects of Acyl Chain Length and Saturation of Diacylglycerols and Phosphatidylcholines on Membrane Monolayer Curvature

The second messenger, diacylglycerol (DAG), introduces negative curvature in phospholipid monolayers and strongly induces the lamellar (L(alpha)) to reverse hexagonal (H(II)) phase transition. The chain lengths and degree of unsaturation of symmetric DAGs influence this effect. Within dioleoylphosphatidylcholine (DOPC) monolayers, the apparent spontaneous radius of curvature (R(0)) of the short, saturated dicaprylglycerol (C10-DCG) itself was determined to be -13.3 A, compared with an R(0) value of -10.1 A for the long, di-monounsaturated dioleoylglycerol (C18-DOG). Such increased length and unsaturation of the DAG acyl chains produces this small change. Di-saturated phosphatidylcholines (PCs) with equal length chains (from C10-C18) with 25 mol % DOG do not form the H(II) phase, even under the unstressed conditions of excess water and alkane. Di-unsaturated PCs with equal chain length (from C14-C18) with 25 mol % DOG do form the H(II) phase. Asymmetric chained PCs (position 1 saturated with varying lengths, position 2 differentially unsaturated with varying lengths) all form the H(II) phase in the presence of 25 mol % DOG. As a general rule for PCs, their unsaturation is critical for the induction of the H(II) phase by DOG. The degree of curvature stress induced by the second messenger DOG in membranes, and any protein that might be affected by it, would appear to depend on chain unsaturation of neighboring PCs.

Curvature and Bending Constants for Phosphatidylserine-Containing Membranes

Phosphatidylserine (PS), an anionic phospholipid of significant biological relevance, forms a multilamellar phase in water with net negative surface charge at pH 7.0. In this study we mixed dioleoylPS (DOPS) with reverse hexagonal (H(II))-forming phosphatidylethanolamine (DOPE), and used x-ray diffraction and osmotic stress to quantify its spontaneous curvature (1/R(0p)) and bending modulus (K(cp)). The mixtures were stable H(II) phases from 5 to 30 mol% PS, providing 16 wt% tetradecane (td) was also added to relieve chain-packing stress. The fully hydrated lattice dimension increased with DOPS concentration. Analysis of structural changes gave an apparent R(0p) for DOPS of +144 A; opposite in sign and relatively flat compared to DOPE (-30 A). Osmotic stress of the H(II) phases did not detect a significantly different bending modulus (K(cp)) for DOPS as compared to DOPE. At pH < or = 4.0, DOPS (with no td) adopted the H(II) phase on its own, in agreement with previous results, suggesting a reversal in curvature upon protonation of the serine headgroup. In contrast, when td was present, DOPS/td formed a lamellar phase of limited swelling whose dimension increased with pH. DOPS/DOPE/td mixtures formed H(II) phases whose dimension increased both with pH and with DOPS content. With tetradecane, estimates put 1/R(0p) for DOPS at pH 2.1 at zero. Tetradecane apparently affects the degree of dissociation of DOPS at low pH.

Elastic properties of polyunsaturated phosphatidylethanolamines influence rhodopsin function

Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (M(II)), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether M(II)-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18 : 0-22 : 6(n - 3)PE, 18 : 0-22 : 5(n)- 6PE and the model lipid 18 : 1(n - 9)-18 : 1,(n- 9)PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 angstroms. In membranes, all three PEs generate high negative curvature elastic stress that shifts the equilibrium of MI(I)/M(II) photointermediates of rhodopsin towards M(II) formation.

Interactions of liposome bilayers composed of 1,2-diacyl-3-succinylglycerol with protons and divalent cations

Bilayer liposomes were prepared by using pure DOSG (1,2-dioleoyl-3-succinylglycerol) or DPSG (1,2-dipalmitoyl-3-succinylglycerol) at pH 7.4 or above. These liposomes undergo destabilization upon incubation with acid. When calcein was used as an entrapped aqueous marker, half maximal content leakage was observed between pH 5.8-6.3. Differential scanning calorimetry showed that at pH 7.4, the chain-melting temperature (Tm) of DPSG was 60.4 degrees C, and increased with decreasing pH (Tm = 57.0 degrees C and 62.7 degrees C at pH 8.9 and 6.7, respectively). Below pH 6.7, extensive phase separation occurred as the major chain melting peak split into three peaks. These three peaks coalesced into one peak below pH 5. Freeze fracture electron micrographs of DOSG liposomes at pH 4 showed the formation of non-bilayer as well as hexagonal phase structures. The effects of divalent cations, such as Ca2+ and Mg2+, on the destabilization of DASG bilayers have also been studied. Differential scanning calorimetry studies of bilayers composed of DPSG showed that both Ca2+ and Mg2+ could increase the Tm of DPSG with increasing concentrations. However, under identical conditions Mg2+ was more effective than Ca2+ in increasing the Tm of DPSG. X-ray diffraction indicated that both Ca2+ and Mg2+ could induce DPSG bilayers to undergo a complete lamellar to hexagonal phase transition. There was a size-dependency on the plasma stability of DOSG liposomes. DOSG liposomes that were smaller in size were more stable in plasma than the larger ones. After incubation with plasma, DOSG liposomes became less acid-sensitive. DOSG immunoliposomes entrapping diphtheria toxin A chain were used as a model for cytoplasmic delivery of the novel pH-sensitive liposomes. The delivery activity was comparable to that of the conventional pH-sensitive liposomes containing unsaturated phosphatidylethanolamine. Our data indicate that the mechanism of liposome destabilization involves extensive bilayer phase separation as well as the formation of non-bilayer structures.