Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.
Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.
复制标题
DOI：
10.1021/bi961876e
复制
发表时间：
1997-01
期刊：
影响因子：
2.9
通讯作者：
D. P. Rogers;Christie G. Brouillette;Jeffrey A. Engler;S. W. Tendian;Linda M. Roberts;V. Mishra;G. Anantharamaiah;S. Lund-Katz;M. C. Phillips;M. J. Ray
中科院分区：
文献类型：
--
作者：
D. P. Rogers;Christie G. Brouillette;Jeffrey A. Engler;S. W. Tendian;Linda M. Roberts;V. Mishra;G. Anantharamaiah;S. Lund-Katz;M. C. Phillips;M. J. Ray
为了研究载脂蛋白hA-I（apo hA-I）第3外显子编码的氨基酸在结构和功能上的作用，利用细菌表达系统制备了人载脂蛋白hA-I（apo hA-I）氨基端缺失突变体（残基1-43）。通过表面活性、与棕榈酰油酰磷脂酰胆碱（POPC）囊泡的脂质结合以及与血浆脂蛋白的脂质结合评估，载脂蛋白δ（1-43）A-I的脂质结合和载脂蛋白hA-I的脂质结合非常相似。初步动力学测量似乎表明，卵磷脂：胆固醇酰基转移酶（LCAT）与突变体的反应性略有下降相比，野生型载脂蛋白hA-I。总的来说，这些结果表明N-末端区域对于LCAT的脂质结合或活化不是必需的。相比之下，通过变性剂诱导的解折叠、荧光探针1-苯胺基萘-8-磺酸盐的结合、表面平衡测量以及远紫外和近紫外圆二色性光谱判断，无脂质apo delta（1-43）A-I和apo hA-I之间存在显著的结构差异。所有光谱和物理测量表明，载脂蛋白δ（1-43）A-I具有折叠的三级结构，尽管它的稳定性明显低于载脂蛋白hA-I。可以得出结论，N-末端43个残基是载脂蛋白hA-I的无脂质构象状态的重要结构元件，与天然载脂蛋白hA-I中的那些残基相比，其缺失诱导了剩余羧基末端残基的根本不同的折叠。
An amino-terminal deletion mutant (residues 1-43) of human apolipoprotein A-I (apo hA-I) has been produced from a bacterial expression system to explore the structural and functional role of these amino acids, encoded by exon 3, in apo hA-I. Lipid binding of apo delta (1-43)A-I and lipid binding of apo hA-I are very similar as assessed by surface activity, lipid association with palmitoyloleoylphosphatidylcholine (POPC) vesicles, and lipid association with plasma lipoproteins. Preliminary kinetic measurements appear to show that the reactivity of lecithin:cholesterol acyltransferase (LCAT) with the mutant is slightly decreased compared to wild-type apo hA-I. Collectively, these results indicate that the N-terminal region is not necessary for lipid binding or activation of LCAT. In contrast, there are significant structural differences between lipid-free apo delta (1-43)A-I and apo hA-I, as judged by denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements, and far- and near-ultraviolet circular dichroic spectroscopy. All spectral and physical measurements indicate apo delta (1-43)A-I has a folded, tertiary structure, although it is significantly less stable than that of apo hA-I. It is concluded that the N-terminal 43 residues are an important structural element of the lipid-free conformational state of apo hA-I, the absence of which induces a fundamentally different fold for the remaining carboxy-terminal residues, compared to those in native apo hA-I.