Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4.
Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4.
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DOI：
10.1021/bi300672s
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发表时间：
2012-07-17
期刊：
影响因子：
2.9
通讯作者：
Saito H
中科院分区：
文献类型：
--
作者：
Mizuguchi C;Hata M;Dhanasekaran P;Nickel M;Phillips MC;Lund-Katz S;Saito H
载脂蛋白（apo）E被认为在与脂质结合时在N-末端螺旋束结构域发生构象变化，从而调节其受体结合活性。本研究采用芘马来酰亚胺（pyrene maleimide）或丙烯酰二氢吡喃（acrylodan）标记apoE 4的N端（S94）和C端（W264或S290）螺旋结构域，研究了N端和C端结构域的构象组织和脂质结合行为。胍变性实验丙烯酰荧光监测表明，组织较少，更多的溶剂暴露的结构的C-末端螺旋相比，N-末端螺旋束。芘激基缔合物荧光和凝胶过滤色谱表明，通过apoE 4的C-末端螺旋存在广泛的分子间螺旋-螺旋接触。芘标记的载脂蛋白E4与卵磷脂小单层囊泡结合后芘荧光增加的比较表明了两步脂质结合过程;载脂蛋白E4最初通过C-末端螺旋与脂质表面结合，随后是N-末端螺旋束结构域的较慢构象重组。与此一致，从色氨酸残基的荧光共振能量转移测量到acryloydan连接在位置94表明，在结合到脂质表面，开放的N-末端螺旋束发生在相同的速率增加芘荧光的N-末端结构域。这种apoE 4的脂质结合的两步机制可能适用于大多数磷脂覆盖的脂蛋白，如VLDL。然而，监测芘荧光结合HDL 3表明，不仅apoE-脂质相互作用，而且蛋白质-蛋白质相互作用是重要的apoE 4结合HDL 3。
Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipids-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL3 suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL3.
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影响因子：
2.9
作者：
Nguyen D;Dhanasekaran P;Nickel M;Nakatani R;Saito H;Phillips MC;Lund-Katz S
通讯作者：
Lund-Katz S
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通讯作者：
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