藻胆体核亚基ApcD定点突变及体内重组功能研究

Site-directed Mutagenesis of ApcD of Core Subunit and Their Spectral Study

在对Anabaena sp.PCC7120藻胆体核亚基ApcD结合色素PCB的体内重组中,发现色素蛋白在提纯前后最大吸收峰和荧光峰发生了红移,从提纯前的605nm及633nm变为提纯后的650nm及665nm.为了研究该现象的原因,构建了ApcD的8个突变体,重组结果显示:突变体ApcD(Y88I)色素蛋白在提纯后的吸收光谱和荧光光谱较提纯前均多出一个峰,分别为668nm,690nm;ApcD(W59Q)、ApcD(Y73A)、ApcD(W87E)色素蛋白在提纯前后的吸收光谱和荧光光谱一致;ApcD(M126S)、ApcD(Y116S)、ApcD(M160T)色素蛋白在提纯前后的吸收光谱一致,而提纯后的荧光峰位置较提纯前分别红移了5nm、7nm和10nm;ApcD(M115I)色素蛋白在提纯前后的吸收光谱和荧光光谱均发生了红移,从提纯前的605nm和633nm变为提纯后的638nm和655nm.这些色素蛋白在酸性尿素溶液变性条件下的最大吸收峰始终在662nm,表明辅基色素仍然是藻蓝胆素;在对PCB-ApcD、PCB-ApcD(Y116S)及PCB-ApcD(M160T)的圆二色谱分析发现,该两个氨基酸的突变均对脱辅基蛋白所连接的色素构象产生了一定的影响,而对重组蛋白的二级结构没有影响.

Absorption and fluorescence spectra of the chromoprotein changed during the ApcD of core subunit from Anabaena sp. PCC 7120 bound PCB with reconstitution in E. coli, the λmax of absorption and fluorescence spectra were 605 nm and 633 nm before purification, while the λmax of absorption and fluorescence spectra were 650 nm and 665 nm after purification. To study the phenomenon above, eight mutants were constructed. It is indicated with reconstitution in E. coli that: the mutant ApcD (Y88I) had one more absor...