Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
Biophys J. 2010 Jul 21;99(2):526-33. doi: 10.1016/j.bpj.2010.04.052.
Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mbetaCD can be measured. Comparison of how cholesterol and CTL partitioning between mbetaCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mbetaCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.
胆固醇在不同的细胞膜隔室之间分布不均匀，胆固醇的含量从内层双层膜向质膜增加。有人认为这种胆固醇梯度对于跨膜蛋白的分拣很重要。胆固醇还被证明在真核细胞膜的侧向组织中起着重要作用。在这项研究中，目的是确定跨膜蛋白如何影响磷脂双层中胆固醇的侧向分布。通过研究设计的模拟蛋白质跨膜螺旋的肽与不同组成的双层膜相互作用，可以了解这一点。为此，我们开发了一种测定法，其中可以测量荧光胆固醇类似物 CTL 在 LUVs 和 mbetaCD 之间的分配。比较 CTL 与具有不同组成的 mbetaCD 和磷脂双层之间的分配情况表明，CTL 类似物可以感知双层组成的变化。因此，CTL 获得的结果可用于了解脂质双层中的胆固醇分布。测量了 WALP23 对 DMPC 双层和 mbetaCD 之间 CTL 分配的影响。结果清楚地表明，WALP23 以浓度依赖的方式增加了双层的有序性（如 CTL 和 DPH 各向异性所见）和甾醇与双层的亲和力。尽管 WALP23 也增加了 DLPC 和 POPC 双层的有序性，但这些脂质对 CTL 分配的影响要小得多。这表明蛋白质对具有较长和饱和酰链的磷脂与甾醇相互作用的影响最大。KALP23 对磷脂双层中酰链有序性没有显著影响，并且 KALP23 包含在 DMPC 双层中略微降低了 CTL 分配到双层中的量。这表明跨膜蛋白可以降低和增加甾醇与周围蛋白质的脂质双层的亲和力。这可能会影响双层内的甾醇分布，从而影响生物膜中的侧向组织。
