STRUCTURAL BASIS OF THE INTERACTIONS OF APOLIPOPROTEIN E

描述(改编自申请者摘要)：总体目标是 为了更全面地了解载脂蛋白的结构 (载脂蛋白)E，尤指与蛋白质结合的能力 低密度脂蛋白(LDL)受体和脂质。点突变是 已知会产生载脂蛋白E的异构体，在胆固醇和 甘油三酯运输。一系列物理-生化技术将被 与载脂蛋白E一起使用，并在大肠杆菌中表达，以解决3个特定的问题 目标。1)了解与磷脂(PL)的相互作用如何改变 构象载脂蛋白E3，使其能与低密度脂蛋白受体结合。这个 脂蛋白相关的赖氨酸(K)残基的微环境， 两亲性，a-螺旋反映螺旋之间的相互作用，因此pKa 在PL蛋白盘状颗粒中的每个13C标记的K残基将是 用(~1H，~(13)C)-异核多量子相干核磁共振确定。这个 需要检验的假设是，由基因诱导的a-螺旋长度和间距 与脂质的相互作用是实现高亲和力结合的关键 低密度脂蛋白受体。2)了解负责的机制 不同大小血清脂蛋白载脂蛋白E亚型亲和力的差异 颗粒，表征载脂蛋白E结合的热力学参数 以及一系列定义大小的脂质颗粒的工程变体将 使用荧光光谱和滴定量热法进行测量。3) 为了了解低密度脂蛋白受体结合域的微环境 与载脂蛋白E分子的结合能有关， K143和K146与受体直接相关的微环境 结合将使用载脂蛋白E在其自由能为 与受体的结合差异很大。受体结合的变化 结构域将由apo E分子的突变或 相关脂质的变化。总体而言，实现这三个目标将 生成关于载脂蛋白E 结构和多态影响蛋白质的功能性质 在生理和病理条件下。

DESCRIPTION (Adapted from Applicant's Abstract): The overall objective is to gain a more complete understanding of the structure of apolipoprotein (apo) E, especially as it relates to the ability of the protein to bind to the low density lipoprotein (LDL) receptor and lipids. Point mutations are known to give isoforms of apo E that function abnormally in cholesterol and triglyceride transport. A range of physical-biochemical techniques will be used with apo E engineered and expressed in E. coli to address 3 specific aims. 1) To understand how interaction with phospholipid (PL) changes the conformation of apo E3 so that it can bind to the LDL receptor. The microenvironments of the lysine (K) residues in lipoprotein-associated, amphipathic, a-helices reflect interactions between helices so the pKa of each 13C-labeled K residue in PL-protein discoidal particles will be determined by (1H, 13C)-heteronuclear multiple quantum coherence NMR. The hypothesis to be tested is that the a-helix lengths and spacings induced by interaction with the lipid are critical for achieving high affinity binding to the LDL receptor. 2) To understand the mechanisms responsible for the differing affinities of apo E isoforms for variously-sized serum lipoprotein particles, the thermodynamic parameters characterizing the binding of apo E and engineered variants to a range of lipid particles of defined sizes will be measured using fluorescence spectroscopy and titration calorimetry. 3) To understand how the microrenvironment in the LDL receptor- binding domain of the apo E molecule correlates with the energetics of binding, the microenvironments of K143 and K146 which are directly involved in receptor binding will be investigated using apo E in states where its free energy of binding to the receptor varies widely. Changes in the receptor binding domain will be induced by either mutagenesis of the apo E molecule or changes in the associated lipid. Overall, achievement of these 3 aims will generate novel quantitative information about the ways in which apo E structure and polymorphism affect the functional properties of the protein in both physiological and pathological conditions.