Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe
Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe
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DOI：
10.1016/s0167-4838(01)00317-x
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发表时间：
2002-02-11
期刊：
影响因子：
--
通讯作者：
Jonas, A
中科院分区：
文献类型：
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作者：
Agree, AKB;Tricerri, MA;Jonas, A
载脂蛋白a - i （apoA-I）作为HDL的一个组成部分，在逆向胆固醇运输中具有重要的生理作用；然而，apoA-I也以低脂或无脂形式存在，它们是HDL代谢的关键中间体和细胞脂质受体。本研究的目的是研究无脂apoA-I的中心和c端区域的结构和稳定性。为此，构建并在大肠杆菌中表达了5个Cys突变体：V119C、A124C、A154C、A190C和A232C。这些突变体用6-丙烯酰-2-二甲氨基萘（acrylodan）特异性标记。并通过CD光谱和各种荧光方法进行了检测。结果表明，Cys残基的引入及其与AC的共价标记并未影响apoA-I的整体结构和稳定性。然而。AC荧光性质揭示了apoA-I的中心和c端半部分的不同片段具有不同的折叠和稳定性。从荧光能量转移数据中，得到了含有色氨酸残基的n端区域与各个AC位置之间的平均距离。当前的结果。结合先前发表的观察结果，构建了无脂apoA-I折叠的三维模型。(C) 2002 Elsevier Science B.V.版权所有
Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan. AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However., AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained. The current results. together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I. (C) 2002 Elsevier Science B.V. All rights reserved.