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

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.