Structure and stability of apolipoprotein A-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation
Structure and stability of apolipoprotein A-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation
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DOI：
10.1021/bi051669r
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发表时间：
2006-01-31
期刊：
影响因子：
2.9
通讯作者：
Atkinson, D
中科院分区：
文献类型：
--
作者：
Gorshkova, IN;Liu, T;Atkinson, D
为了鉴定载脂蛋白A-I（apoA-I）中心区域中对蛋白质结构和稳定性重要的残基和片段，我们研究了D102 A [D103 A，E110 A/E111 A，R116 V/K118 A和R160 V/H162 A]四种双电荷切除以及Delta（61-78）和Delta（61-78）两种缺失突变对apoA-I的影响（121 - 142），对apoA-I在无脂质状态和重构的盘状磷脂-胆固醇-apoA-I颗粒（rHDL）中的构象和稳定性的研究。结果表明，位于第4螺旋和第61 ~ 78位残基之间的D102/D103和E110/E111参与了apoA-I在无脂状态和rHDL中构象和稳定性的维持。位于螺旋4的R116/K118对apoA-I在rHDL中的构象和稳定性是必需的，但对蛋白质的无脂质状态不是至关重要的。螺旋6中的R160 V/H162 A取代导致无脂质apoA-I的三级结构不那么紧凑，而对无脂质或脂质结合的二级构象没有显著影响，这表明R160/H162参与了重要的螺旋间相互作用。A（121 - 142）突变体的结果，以及我们早期的发现，提示在无脂质的apoA-I中，残基121和143之间的主要片段的无序结构，可能包括残基131-143。我们的研究结果提供了第一个实验证据的稳定rHDL的特定静电螺旋间的相互作用，在协议的双带模型。改变的构象和稳定性的apoA-I突变体在体外和体内的apoA-I和脂质稳态的功能的影响进行了讨论。
To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A[D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121 -142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the A(121 -142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.