Mi Wei, Li Lanfen, Su Xiao-Dong
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, No. 5 Yi Heyuan Road, Beijing 100871, China.
Biochem Biophys Res Commun. 2008 Apr 18;368(4):919-22. doi: 10.1016/j.bbrc.2008.02.021. Epub 2008 Feb 14.
Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys(114), and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.
人类氯离子细胞内通道蛋白2（CLIC2）的结构研究一直受到难以获得合适晶体这一问题的阻碍，主要原因是该蛋白质含有暴露的半胱氨酸。使用了几种化学试剂与CLIC2上的半胱氨酸反应，以改变蛋白质的氧化还原状态。通过用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）处理蛋白质，我们获得了高质量的晶体，在家庭X射线源下衍射分辨率优于2.5埃。解析CLIC2的晶体结构后，我们发现DTNB与Cys(114)发生了反应，并使CLIC2处于均匀的氧化状态。这项研究表明，DTNB修饰极大地改善了CLIC2的结晶，这意味着该方法可能总体上对其他含有暴露半胱氨酸的蛋白质也有用。
