The Association-Dissociation Behavior of the ApoE Proteins: Kinetic and Equilibrium Studies
The Association-Dissociation Behavior of the ApoE Proteins: Kinetic and Equilibrium Studies
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DOI：
10.1021/bi101407m
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发表时间：
2010-11-09
期刊：
影响因子：
2.9
通讯作者：
Frieden, Carl
中科院分区：
文献类型：
--
作者：
Garai, Kanchan;Frieden, Carl
载脂蛋白E家族包括三种主要的蛋白质亚型：载脂蛋白E4(ApoE4)、载脂蛋白E3和载脂蛋白2。这些亚型含有299个残基，只有单一氨基酸的变化不同，但在这三种亚型中，只有载脂蛋白E4是阿尔茨海默病的风险因素。在微摩尔浓度下，无脂ApoE主要以四聚体形式存在。在较稀的溶液中，低分子质量的物种占主导地位。利用荧光相关光谱(FCS)、分子间荧光共振能量转移(FRET)和沉淀法，我们发现ApoE的缔合-解离反应可以用单体-二聚体-四聚体过程来模拟。通过测定ApoE的解离动力学，测定了ApoE的缔合和解离速率常数，并与平衡常数进行了比较。分子间FRET测定的解离动力学显示出两个阶段，反映了四聚体到二聚体和二聚体到单体的解离，其中四聚体到二聚体的解离比二聚体到单体的解离更快。不同的ApoE亚型的速率常数不同，表明缔合-解离过程是异构型的。值得注意的是，对于扩散控制的过程，缔合速率常数几乎比预期慢2个数量级。在丙烯酰胺存在下，用色氨酸荧光法监测了解离动力学，发现数据符合单体-二聚体-四聚体模型。该方法结合多种方法建立了载脂蛋白E自缔合反应模式。
The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer's disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association-dissociation reaction of ApoE can be modeled with a monomer-dimer-tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association-dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer-dimer-tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.