Fastenberg Michael E, Shogomori Hidehiko, Xu Xiaolian, Brown Deborah A, London Erwin
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
Biochemistry. 2003 Oct 28;42(42):12376-90. doi: 10.1021/bi034718d.
Sphingolipid/cholesterol-rich rafts are membrane domains thought to exist in the liquid-ordered state. To understand the rules governing the association of proteins with rafts, the behavior of a model membrane-inserted hydrophobic polypeptide (LW peptide, acetyl-K(2)W(2)L(8)AL(8)W(2)K(2)-amide) was examined. The distribution of LW peptide between coexisting ordered and disordered lipid domains was probed by measuring the amount of LW Trp fluorescence quenched by a nitroxide-labeled phospholipid that concentrated in disordered lipid domains. Strong quenching of the Trp fluorescence (relative to quenching in model membranes lacking domains) showed that LW peptide was concentrated in quencher-rich disordered domains and was largely excluded from ordered domains. Exclusion of LW peptide from the ordered domains was observed both in the absence and in the presence of 25-33 mol % cholesterol, indicating that the peptide is relatively excluded both from gel-state domains (which form in the absence of cholesterol) and from liquid-ordered-state domains (which form at high cholesterol concentrations). Because exclusion was also observed when ordered domains contained sphingomyelin in place of DPPC, or ergosterol in place of cholesterol, it appeared that this behavior was not strongly dependent on lipid structure. In both the absence and the presence of 25 mol % cholesterol, exclusion was also not strongly dependent upon the fraction of the bilayer in the form of ordered domains. To evaluate LW peptide behavior in more detail, an analysis of the effects of domain size and edges upon quenching was formulated. This analysis showed that quenching can be affected both by domain size and by whether a fluorescent molecule localized at domain edges. Its application to the quenching of LW peptide indicated that the peptide did not preferentially reside at the boundaries between ordered and disordered domains.
富含鞘脂/胆固醇的脂筏是被认为以液晶态存在的膜结构域。为了理解蛋白质与脂筏结合的规律，研究了一种模拟膜插入疏水多肽（LW肽，乙酰基-K(2)W(2)L(8)AL(8)W(2)K(2)-酰胺）的行为。通过测量被集中在无序脂质结构域的氮氧化物标记磷脂淬灭的LW色氨酸荧光量，探究了LW肽在共存的有序和无序脂质结构域之间的分布。色氨酸荧光的强烈淬灭（相对于缺乏结构域的模型膜中的淬灭）表明LW肽集中在富含淬灭剂的无序结构域中，并且在很大程度上被排除在有序结构域之外。在不存在和存在25 - 33 mol%胆固醇的情况下，均观察到LW肽被排除在有序结构域之外，这表明该肽相对被排除在凝胶态结构域（在无胆固醇时形成）和液晶态结构域（在高胆固醇浓度时形成）之外。由于当有序结构域含有鞘磷脂代替二棕榈酰磷脂酰胆碱（DPPC），或麦角固醇代替胆固醇时也观察到了排除现象，所以这种行为似乎并不强烈依赖于脂质结构。在不存在和存在25 mol%胆固醇的情况下，排除现象也不强烈依赖于以有序结构域形式存在的双层膜的比例。为了更详细地评估LW肽的行为，制定了对结构域大小和边缘对淬灭影响的分析。该分析表明，淬灭可受结构域大小以及荧光分子是否定位于结构域边缘的影响。将其应用于LW肽的淬灭表明，该肽并非优先位于有序和无序结构域之间的边界处。
