Zlobin Nikolai, Evlakov Konstantin, Alekseev Yakov, Blagodatskikh Konstantin, Babakov Aleksei, Taranov Vasiliy
All-Russia Research Institute of Agricultural Biotechnology, Russian Academy of Sciences, Russia.
Biochem Biophys Rep. 2016 Feb 9;5:502-508. doi: 10.1016/j.bbrep.2016.02.004. eCollection 2016 Mar.
Plant cold shock domain proteins (CSDP) participate in maintenance of plant stress tolerance and in regulating their development. In the present paper we show that two out of three extremophyte plant proteins EsCSDP1-3, namely EsCSDP1 and EsCSDP3, possess high DNA-melting activity. DNA-melting activity of proteins was evaluated using molecular beacon assay in two ways: by measuring Tm parameter (the temperature at which half of the DNA beacon molecules is fully melted) and the beacon fluorescence at 4 °C. As the ratio protein/beacon was increased, a decrease in Tm was observed. Besides DNA-melting activity of full proteins, activity was measured for three isolated cold shock domains EsCSD1-3, C-terminal domain of EsCSDP1 (EsZnF1), as well as a mixture of EsCSD1 and EsZnF1. The Tm reduction efficiency of proteins formed the following sequence: EsCSDP3≈EsCSDP1>(EsCSD1+EsZnF1)>EsZnF1>EsCSDP2. Only full proteins EsCSDP3 and EsCSDP1 demonstrated DNA-melting activity at 4 °C. The presented experimental data indicate that i: interaction of EsCSDP1-3 with beacon single-stranded region is obligatory for efficient melting; ii: cold shock domain and C-terminal domain with zinc finger motifs should be present in one protein molecule to have high melting activity.
植物冷休克结构域蛋白（CSDP）参与维持植物的胁迫耐受性并调节其发育。在本文中，我们表明三种极端植物蛋白EsCSDP1 - 3中的两种，即EsCSDP1和EsCSDP3，具有高DNA解链活性。使用分子信标分析法通过两种方式评估蛋白质的DNA解链活性：测量Tm参数（DNA信标分子一半完全解链时的温度）和4℃下的信标荧光。随着蛋白质/信标比例的增加，观察到Tm降低。除了完整蛋白质的DNA解链活性外，还测量了三个分离的冷休克结构域EsCSD1 - 3、EsCSDP1的C末端结构域（EsZnF1）以及EsCSD1和EsZnF1的混合物的活性。蛋白质的Tm降低效率形成以下序列：EsCSDP3≈EsCSDP1>(EsCSD1 + EsZnF1)>EsZnF1>EsCSDP2。只有完整蛋白质EsCSDP3和EsCSDP1在4℃下表现出DNA解链活性。所呈现的实验数据表明：i：EsCSDP1 - 3与信标单链区域的相互作用对于有效解链是必需的；ii：冷休克结构域和具有锌指基序的C末端结构域应存在于一个蛋白质分子中以具有高解链活性。
