Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Biochemistry. 2010 Nov 9;49(44):9533-41. doi: 10.1021/bi101407m.
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.
载脂蛋白 E 家族由三种主要的蛋白质异构体组成：载脂蛋白 E4（ApoE4）、ApoE3 和 ApoE2。这三种异构体都含有 299 个残基，仅在单个氨基酸上有所不同，但只有 ApoE4 是阿尔茨海默病的风险因素。在微摩尔浓度下，无脂载脂蛋白 E 主要以四聚体形式存在。在更稀的溶液中，低分子量的物质占优势。使用荧光相关光谱（FCS）、分子间荧光共振能量转移（FRET）和沉降方法，我们发现载脂蛋白 E 的缔合-解离反应可以用单体-二聚体-四聚体过程来模拟。平衡常数是从沉降数据中确定的，而单体和二聚体的缔合和解离的个别速率常数是通过测量载脂蛋白 E 解离的动力学来确定的，与平衡常数一致。通过分子间 FRET 测量的解离动力学显示出两个相，反映了四聚体到二聚体和二聚体到单体的解离，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 异构体的速率常数不同，表明缔合-解离过程是异构体特异性的。引人注目的是，缔合速率常数比扩散控制过程预期的慢近 2 个数量级。在丙烯酰胺存在的情况下，也通过色氨酸荧光监测解离动力学，数据与单体-二聚体-四聚体模型一致。结合多种方法的方法确定了载脂蛋白 E 自组装的反应方案。
