Ya-Wei Hsueh

The Effect of Ceramide on Phosphatidylcholine Membranes: A Deuterium NMR Study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cers chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.

Effect of Sterol Structure on the Physical Properties of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine Membranes Determined Using 2H Nuclear Magnetic Resonance

The effect of a series of phytosterols on lipid chain ordering in 1-palmitoyl((2)H31)-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) multibilayer vesicles was examined by (2)H NMR spectroscopy at 25 °C. These results, along with existing data for other sterols, indicate that the ordering power of sterols in POPC-d31 depends on subtle aspects of sterol structure. Cholesterol, 7-dehydrocholesterol (7-DHC), campesterol, β-sitosterol, ergosterol, brassicasterol, and stigmasterol all increase the lipid chain order as sterol concentration is increased. However, saturation of the ordering occurs at different sterol concentrations for ergosterol (as previously reported), brassicasterol, β-sitosterol, and stigmasterol. Here our interest lies in finding which part of the sterol structure is responsible for the observed saturation of the palmitoyl chain order as a function of sterol concentration. In particular, we propose that the saturation of the ordering of POPC-d31/brassicasterol and POPC-d31/stigmasterol membranes at quite low sterol concentrations is due to the presence of a double bond at C22. We also discuss how the structural differences between the sterols affect their ability to intercalate between the POPC acyl chains. Furthermore, the effective solubility of sterols in POPC is discussed in relation to the dependence of maximum POPC-d31 chain order vs sterol concentration.

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[(2)H(31)]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

Abstract To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C6-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by 2H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C6-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel + fluid phase coexistence. Inclusion of both fluorescent probes (1 mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C6-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.