Davidson W S, Hazlett T, Mantulin W W, Jonas A
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, USA.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13605-10. doi: 10.1073/pnas.93.24.13605.
Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apo AI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI delta 44-126, apoAI delta 139-170, and apoAI delta 190-243). Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI delta 44-126 was 27% while the other mutants and native apoAI averaged 55 +/- 2%, suggesting that the missing N-terminal portion contains most of the alpha-helical structure of lipid-free apoAI. ApoAI delta 44-126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for alpha-helix stability in the lipid-free state, whereas the C terminus is required for alpha-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in alpha-helical content observed when native apoAI binds lipid results from the formation of alpha-helix primarily in the C-terminal half of the molecule.
载脂蛋白AI（apoAI）是高密度脂蛋白的主要蛋白质成分，在人体胆固醇稳态中起关键作用；然而，apoAI的结构尚未完全清楚。为了验证apoAI是由结构域组成的这一假说，对在大肠杆菌中表达的人apo AI的三个缺失突变体进行了溶液中和重组高密度脂蛋白颗粒中的研究。每个突变体都缺少三个特定区域中的一个，这三个区域共同涵盖了天然apoAI几乎整个243个氨基酸序列（apoAI δ44 - 126、apoAI δ139 - 170和apoAI δ190 - 243）。圆二色光谱显示，无脂apoAI δ44 - 126的α-螺旋含量为27%，而其他突变体和天然apoAI的平均含量为55±2%，这表明缺失的N端部分包含无脂apoAI的大部分α-螺旋结构。apoAI δ44 - 126在脂质结合后α-螺旋增加幅度最大（增加125%，而其他平均增加25%），证实了apoAI C端一半在脂质结合中的重要性。变性研究表明，apoAI的N端一半主要负责无脂状态下α-螺旋的稳定性，而C端对于脂质结合时α-螺旋的稳定性是必需的。我们得出结论，apoAI的N端一半（氨基酸44 - 126）负责无脂apoAI的大部分α-螺旋结构和边缘稳定性，而C端（氨基酸139 - 243）结构较松散。天然apoAI结合脂质时观察到的α-螺旋含量增加主要是由于分子C端一半形成了α-螺旋。
