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首页> 外文期刊>Biochemistry >Lipid-binding studies of human apolipoprotein a-I and its terminally truncated mutants.
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Lipid-binding studies of human apolipoprotein a-I and its terminally truncated mutants.

机译：人载脂蛋白A-I及其末端截断突变体的脂质结合研究。

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Apolipoprotein A-I (apoA-I, 243 amino acids) is the major protein of high-density lipoproteins (HDL) that plays an important structural and functional role in lipid transport and metabolism. The central region of apoA-I (residues 60-183) is predicted to contain exclusively amphipathic alpha-helices formed from tandem 22-mer sequence repeats. To analyze the lipid-binding properties of this core domain, four terminally truncated mutants of apoA-I, Delta(1-41), Delta(1-59), Delta(1-41,185-243), and Delta(1-59,185-243), were expressed in baculovirus infected Sf-9 cells. The effects of mutations on the ability of apoA-I to form bilayer disk complexes with dimyristoyl phosphatidylcholine (DMPC) that resemble nascent HDL were analyzed by density gradient ultracentrifugation and electron microscopy (EM). The N-terminal deletion mutants, Delta(1-41) and Delta(1-59), showed altered lipid-binding ability as compared to plasma and wild-type apoA-I, and in the double deletion mutants, Delta(1-41, 185-243) and Delta(1-59, 185-243), the lipid binding was abolished. Thermal unfolding of variant apoA-I/DMPC complexes monitored by circular dichroism (CD) showed hysteresis and a shift in the melting curves by about -12 degrees C upon reduction in the heating rate from 1.0 to 0.067 K/min. This indicates an irreversible kinetically controlled transition with a high activation energy E(a) = 60 +/- 5 kcal/mol. CD and EM studies of the apoA-I/DMPC complexes at different pH demonstrated that changes in the net charge or in the charge distribution on the apoA-I molecule have critical effects on the conformation and lipid-binding ability of the protein.
机译：载脂蛋白A-I（apoA-1,243氨基酸）是高密度脂蛋白（HDL）的主要蛋白质，其在脂质转运和代谢中起重要的结构和功能作用。预计ApoA-I（残留物60-183）的中心区域被含有由串联22-MEL序列的重复形成的专门形成的两亲性α-螺旋。分析该核心结构域的脂质结合特性，ApoA-1的四个末端截断突变体，Delta（1-41），Delta（1-59），Delta（1-41,185-243）和Delta（1-59,185 -243），在杆状病毒感染的SF-9细胞中表达。通过密度梯度超速离心和电子显微镜（EM）分析突变对具有类似鼻塞磷脂酰胆碱（DMPC）的Divyristoyl磷脂酰胆碱（DMPC）的双层盘络合物的效果。与血浆和野生型apoA-1相比，N-末端缺失突变体，δ（1-41）和δ（1-59）显示出改变的脂质结合能力，以及在双缺失突变体，Delta（1- 41,185-243）和Delta（1-59,185-243），废除了脂质结合。通过圆形二色性（CD）监测的变体ApoA-I / DMPC复合物的热展开显示滞后和熔点曲线的偏移在加热速率从1.0至0.067 k / min的降低时通过约-12℃。这表明具有高激活能量E（a）= 60 +/- 5kcal / mol的不可逆的动力学控制过渡。在不同pH下的APOA-I / DMPC复合物的CD和EM研究证明了净电荷或在APOA-I分子上的电荷分布的变化对蛋白质的构象和脂质结合能力具有统计影响。

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