High DNA melting activity of extremophyte Eutrema salsugineum cold shock domain proteins EsCSDP1 and EsCSDP3.
High DNA melting activity of extremophyte Eutrema salsugineum cold shock domain proteins EsCSDP1 and EsCSDP3.
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DOI：
10.1016/j.bbrep.2016.02.004
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发表时间：
2016-03
影响因子：
2.7
通讯作者：
Taranov V
中科院分区：
文献类型：
--
作者：
Zlobin N;Evlakov K;Alekseev Y;Blagodatskikh K;Babakov A;Taranov V
植物冷激结构域蛋白（CSDP）参与植物抗逆性的维持和发育调控。在本文中，我们表明，两个极端植物植物盐地黄皮蛋白EsCSDP 1 -3，即EsCSDP 1和EsCSDP 3，具有高的DNA解链活性。使用分子信标测定以两种方式评估蛋白质的DNA解链活性：通过测量Tm参数（一半DNA信标分子完全解链的温度）和4 °C下的信标荧光。随着蛋白质/信标比率增加，观察到Tm降低。除了全蛋白的DNA解链活性外，还测量了三个分离的冷休克结构域EsCSD 1 -3、EsCSDP 1的C-末端结构域（EsZnF 1）以及EsCSD 1和EsZnF 1的混合物的活性。各蛋白质的Tm降低效率顺序为：EsCSDP 3> EsCSDP 1>（EsCSD 1 + EsZnF 1）> EsZnF 1> EsCSDP 2。只有全蛋白EsCSDP 3和EsCSDP 1在4 °C下表现出DNA解链活性。实验结果表明：1. EsCSDP 1 -3与信标单链区的相互作用是有效解链的必要条件; 2.具有锌指结构的冷休克结构域和C-末端结构域应存在于一个蛋白分子中，以具有高的解链活性。EsCSDP 1和EsCSDP 3蛋白具有高的DNA解链活性。高的DNA解链活性是由蛋白质分子的N端和C端区域的协同作用决定的。解链活性的表现需要与单链DNA区域的相互作用。
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 Eutrema salsugineum 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. EsCSDP1 and EsCSDP3 proteins possess high DNA-melting activity. High DNA-melting activity is determined by cooperative action of N-terminal and C-terminal regions of protein molecule. Interaction with single-stranded DNA regions is required for melting activity manifestation.