Negi Shigeru, Itazu Masako, Imanishi Miki, Nomura Akiko, Sugiura Yukio
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochem Biophys Res Commun. 2004 Dec 10;325(2):421-5. doi: 10.1016/j.bbrc.2004.10.045.
To investigate the properties of unnatural zinc finger peptides with CysHis(3)-type ligand combinations, the HCHH- and CHHH-type zinc finger proteins (zf(HCHH) and zf(CHHH), respectively) were created by mutating Cys to His in the Cys(2)His(2)-type zinc finger of the transcription factor Sp1 (zf(CCHH)). The CD measurements clearly show that the single-finger CysHis(3)-type zinc finger peptides (zf(HCHH)f2 and zf(CHHH)f2) are folded by complexation with Zn(II). From the gel mobility shift assays, the CysHis(3)-type zinc finger proteins (zf(HCHH) and zf(CHHH)) evidently bind to the GC-box DNA, though the DNA binding affinity is lower than that of the wild CCHH-type zinc finger protein. Furthermore, the zf(HCHH)f2 and zf(CHHH)f2 peptides catalyze the hydrolysis of the 4-nitrophenyl acetate in contrast to the catalytically inactive zf(CCHH) peptide. This is the first study of the CysHis(3)-type zinc finger proteins and also provides useful information for redesigning artificial metalloproteins.
为了研究具有CysHis(3)型配体组合的非天然锌指肽的性质，通过将转录因子Sp1的Cys(2)His(2)型锌指（zf(CCHH)）中的Cys突变为His，构建了HCHH型和CHHH型锌指蛋白（分别为zf(HCHH)和zf(CHHH)）。圆二色性测量清楚地表明，单指CysHis(3)型锌指肽（zf(HCHH)f2和zf(CHHH)f2）通过与Zn(II)络合而折叠。凝胶迁移率变动分析表明，CysHis(3)型锌指蛋白（zf(HCHH)和zf(CHHH)）明显与GC盒DNA结合，尽管其DNA结合亲和力低于野生型CCHH型锌指蛋白。此外，与无催化活性的zf(CCHH)肽相反，zf(HCHH)f2和zf(CHHH)f2肽催化乙酸对硝基苯酯的水解。这是对CysHis(3)型锌指蛋白的首次研究，也为重新设计人工金属蛋白提供了有用信息。
