人载脂蛋白A-Ⅰ半胱氨酸突变体的二级结构和脂质结合能力的比较  

Comparison of Secondary Structure and Capabilities of Lipid Binding Between Different Cysteine Mutants of Apolipoprotein A-Ⅰ

作　　者：祝学卫[1] 吴刚[1] 曾武威[1] 薛红[1] 陈保生[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院医学分子生物学国家重点实验室,北京100005

出　　处：《中国生物化学与分子生物学报》2005年第5期603-609,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究"973"项目资助(G2000056902);国家自然科学基金(No.39970170)资助~~

摘　　要：为探讨人载脂蛋白A-Ⅰ(apoA-Ⅰ,apolipoproteinA-Ⅰ)α螺旋不同位点的半胱氨酸突变后,对蛋白二级结构和脂质结合能力的影响,利用定点诱变技术构建apoA-Ⅰ的天然半胱氨酸突变体apoA-ⅠMilano(R173C),及其它α螺旋片段上的半胱氨酸突变体,分别为apoA-Ⅰ(S52C),apoA-Ⅰ(N74C),apoA-Ⅰ(L107C),apoA-Ⅰ(K129C),和apoA-Ⅰ(L195C).观察比较各种野生型及突变apoA-Ⅰ单体蛋白的α螺旋含量和二级结构稳定性及其脂质结合能力.结果显示,野生型apoA-Ⅰ,apoA-Ⅰ(S52C),apoA-Ⅰ(N74C),apoA-Ⅰ(L107C),apoA-Ⅰ(K129C),apoA-ⅠMilano和apoA-Ⅰ(L195C)的α螺旋含量分别为54±4%,49±4%,50±2%,51±6%,56±4%,52±3%,和54±1%,各种蛋白的α螺旋含量无显著性差异(P>0.05).野生型apoA-Ⅰ的变性标准自由能(ΔG0D)为10.5kJ/mol;apoA-Ⅰ(S52C)和apoA-ⅠMilano的ΔG0D比野生型低2.1kJ/mol;而apoA-Ⅰ(K129C)的ΔG0D比野生型apoA-Ⅰ高1.6kJ/mol.与野生型apoA-Ⅰ相比,apoA-Ⅰ(K129C)和apoA-Ⅰ(L195C)两个突变体与脂质结合能力明显下降(P<0.05),而其它半胱氨酸突变体(包括apoA-ⅠMilano)在脂质结合动力学方面与野生型apoA-Ⅰ无明显差异.以上结果提示,不同位点发生的半胱氨酸突变对apoA-Ⅰ单体蛋白的α螺旋含量无明显影响,但对蛋白的二级结构稳定性和脂质结合能力影响不尽相同.To explore the influences of cysteine mutations at the different sites of helices upon the secondary structural and functional properties of human apolipoprotein A- Ⅰ （apoA- Ⅰ ）, site-directed mutagenesis was employed to construct the natural （apoA- Ⅰ Milano, R173C） or unnatural cysteine mutants of apoA- Ⅰ , named as apoA-Ⅰ （S52C）, apoA-Ⅰ （N74C）, apoA-Ⅰ（L107C）, apoA-Ⅰ （K129C）, and apoA-Ⅰ （L195C）, respectively. For the recombinant proteins, the a helical contents, the secondary structural stability and the capabilities to bind to lipid were investigated, respectively. The results showed that the a helical contents of the monomeric wild type apoA-Ⅰ , apoA-Ⅰ （S52C）, apoA- Ⅰ （N74C）, apon- Ⅰ （L107C）, apoA-Ⅰ（K129C）, and apoA-Ⅰ（L195C）were 54 ± 4%,49 ± 4%,50 ± 2%,51 ± 6%,56 ± 4%,52 ± 3%,and 54 ± 1%, respectively. There were no statistical significances between them （ P 〉 0.05）. The free energy of denaturation （△GD^0） of wild type apoA-Ⅰ was 10.5 kJ/mol. △GD^0 of apoA-Ⅰ（S52C）and apoA-Ⅰ Milano decreased by 2.1 kJ/mol and △GD^0 of apoA-Ⅰ（K129C）increased by 1.6 kJ/mol, when compared with wild type apoA-Ⅰ. Binding capacity of apoA-Ⅰ（K129C） and apoA-Ⅰ（L195C）to lipid exhibited significantly lower affinity lipid than that of wild type apoA-Ⅰ （P 〈 0.05）, whereas there was no significant difference between the other cysteine mutants （including apoA-Ⅰ Milano ） and the wild type （P 〉 0.05） has been observed. These results indicate that the cysteine mutations at the different sites have not significant effect on the α helical content of apoA-Ⅰ, but have different influence on its secondary structural stability and its ability to bind to lipids.

关 键 词：人载脂蛋白A—Ⅰ 半胱氨酸突变体 α螺旋含量 二级结构稳定性 脂质结合能力 

分 类 号：Q71[生物学—分子生物学]