| Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity
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Author:
Date:
2017-02-20
[Abstract] Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.
[摘要] 昼夜节律是显示内源性振荡的生物过程，周期为〜24小时。它们允许生物体预期并准备好主要由地球旋转引起的环境变化。昼夜节律由由蛋白质，DNA和/或RNA组成的昼夜节律钟驱动。蓝藻的昼夜节律时钟是最简单的研究模型之一。它们含有三种时钟蛋白质KaiA，KaiB和KaiC，其可用于体外重组实验和本方案所述的KaiC的自磷酸酶活性的测定。
背景 行星地球的旋转导致〜24小时的昼夜振荡。为了适应并有效地利用环境的这种节奏变化，大多数（如果不是全部）生物体具有约24小时的内源性活动节律，这被称为昼夜节律。昼夜节律为这些生物提供进化优势。昼夜节律的长期破坏是非常有害的（Ma et al。，2013）。在人类中，包括癌症，高血压和睡眠障碍在内的许多疾病与昼夜节律紊乱密切相关（Shi等人，2013; Roenneberg和Merrow，2016）。
＆nbsp;昼夜节律由称为昼夜节律钟的内生节律发生器控制。功能性昼夜节律钟具有三个功能：接受环境信息，将环境提示转变为振荡信号，并将这些信号转发到下游调制器（Pattanayak和Rust，2014）。蓝细菌是具有良好研究的昼夜节律钟的最简单的生物体，其中振荡发生器由三种蛋白质控制：KaiA，KaiB和KaiC（Mackey等人，2011; Johnson＆amp; et al。 ...
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| Analytical Gel Filtration for Probing Heavy Metal Transfer between Proteins
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Author:
Date:
2016-08-05
[Abstract] Heavy metals can cause damage to biomolecules such as proteins and DNA in multiple ways. Cells therefore strive for keeping intracellular (heavy) metal ions bound to specific proteins that are capable of handling detoxification, export or integration as cofactors. Metal binding proteins usually provide specific coordination sites that bind certain ions with ultrahigh affinity, with the thermodynamic driving force being the stability of organometallic complexes. However, the metal binding properties of these proteins can be highly variable. Therefore the transfer of specific ions between separate proteins or even between distinct binding sites located on one and the same protein does not always follow affinity gradients, but depends on particular protein interactions that are difficult to ...
[摘要] 重金属可以以多种方式对生物分子例如蛋白质和DNA造成损害。因此，细胞努力保持细胞内（重）金属离子结合到能够处理解毒，输出或整合作为辅因子的特定蛋白质。金属结合蛋白通常提供结合某些离子的特异性配位位点，具有超高的亲和力，热力学驱动力是有机金属配合物的稳定性。然而，这些蛋白质的金属结合性质可以是高度可变的。因此，在分开的蛋白质之间或甚至在位于同一蛋白质上的不同结合位点之间的特异性离子的转移不总是遵循亲和梯度，而是取决于难以预测的特定蛋白质相互作用。我们建立了一种适合探测两种蛋白质之间的金属转移的方法，只要这些蛋白质可以进行纯化和体外处理。它由金属负载，共孵育和金属交换蛋白的分离，随后确定结合金属含量。该方法通过我们探测膜 - 外在金属结合结构域MBD2和来自大肠杆菌的铜输出ATP酶的跨膜结构域的膜 - 外部金属结合结构域MBD2之间的铜（I）转移的实验数据来举例说明（Drees < em=""> 。，2015）。
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| Substrate Specificity of Recombinant Ser/Thr Protein Kinase
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Author:
Date:
2015-03-20
[Abstract] Protein kinases are enzymes that phosphorylate proteins in a cell. Determination of kinase activity in reactions of phosphorylation is a very convenient way for a biochemical characterization of this group of enzymes. Here we describe a method to determine the activity of a recombinant Ser/Thr protein kinase using as a possible substrate MBP, H1, and BSA.
[摘要] 蛋白激酶是磷酸化细胞中蛋白质的酶。 磷酸化反应中激酶活性的测定是这组酶的生物化学表征的非常方便的方法。 在这里，我们描述了一种使用可能的底物MBP，H1和BSA确定重组Ser/Thr蛋白激酶活性的方法。
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