Bunge Andreas, Müller Peter, Stöckl Martin, Herrmann Andreas, Huster Daniel
Junior Research Group Structural Biology of Membrane Proteins, Institute of Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Biophys J. 2008 Apr 1;94(7):2680-90. doi: 10.1529/biophysj.107.112904. Epub 2008 Jan 4.
A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, (2)H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by (2)H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger (2)H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.
使用荧光显微镜、（2）H NMR和电子顺磁共振光谱对棕榈酰油酰磷脂酰胆碱（POPC）、棕榈酰赤型鞘氨醇磷酸胆碱（PSM）和胆固醇以37.5:37.5:25摩尔/摩尔/摩尔的混合比例组成的三元脂质混合物进行了表征。合成的PSM为研究筏状相的分子性质提供了一个极好的分子。它在311 K的温度下显示出狭窄的相变，并且有带有全氘代sn-2链的市售产品。荧光显微镜显示，在液晶态和液晶无序脂质相的共存区域中观察到混合膜存在较大的不均匀性。在310 K以上，未显示出光学可检测的相分离。通过（2）H NMR和电子顺磁共振实验观察到，随着温度降低，胆固醇重新分布到大型液晶态PSM/胆固醇结构域中，并且液晶无序的POPC结构域中的胆固醇减少。然而，胆固醇并没有完全分离到液晶态相中，并且富含POPC的结构域在相共存区域中也含有甾醇。我们进一步比较了在313 K（即液晶相状态）下筏状混合物中氘代PSM或POPC的序参量和堆积性质。PSM在筏状相中显示出比POPC明显更大的（2）H NMR序参量。这可以通过脂质种类之间胆固醇的不均匀相互作用以及磷脂彼此之间的相互影响来解释。这些观察结果表明，在313 K的液晶相中脂质也存在不均匀分布。根据在完全均匀混合的膜中序参量相同这一前提，我们可以确定在所有脂质的主相变温度以上的PSM/POPC/胆固醇混合物中最小微区尺寸为45 - 70 nm。
