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Lithium potassium acetyl phosphate

乙酰磷酸锂钾

公司名称: Sigma-Aldrich
产品编号: A0262
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Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
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Date:
2017-08-20
[Abstract]  We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in ... [摘要]  我们已经开发了产生组氨酸激酶DesK及其同源反应调节物DesR的位点特异性变体的方案,有助于捕获蛋白质的不同信号状态。两个合作伙伴在大肠杆菌中的共表达,确保调节剂过量,对于DesK:DesR复合物的可溶性生产和进一步纯化是至关重要的。通过使用分子置换的X射线晶体学解决了捕获在磷酸转移酶和磷酸酶反应步骤中的复合物的3D结构。该解决方案不是微不足道的,我们发现在用作搜索探针的硅片生成的模型中,有助于将大部分复合物放置在不对称单元中。电子密度图就足够清楚了,可以进行人工建模,获得完整的原子模型。这些方法有助于解决细菌信号领域的主要挑战,即获得稳定的激酶:调节复合物,具有不同的构象状态,适用于高分辨率晶体学研究。
【背景】关于细菌信号复合物,特别是双组分系统(TCS)的结构信息仍然很少(Casino et al。,2009; Gao and Stock,2009)。 TCS包含几乎所有细菌中的感觉组氨酸激酶(HK)和响应调节剂(RR)配偶体,它们允许细胞感知环境并通过适应性反应相应地反应。尽管在信号传输中这种切换机制的重要性(Trajtenberg等,2016),结构信息对于采用不同功能状态的TCS复合体甚至更为有限。我们研究了DesK-DesR途径(de Mendoza,2014),一种来自枯草芽孢杆菌的TCS,其参与调节细胞膜组成以适应降低双层流动性的线索,如冷休克。 ...

Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro
Author:
Date:
2013-11-20
[Abstract]  Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an aspartate residue (Boulanger et al., 2013). This method is based on the proprietary adduct, Phos-tagTM, a dinuclear metal complex, which together with Zn2+ or Mn2+, forms a complex with a phosphomonoesterdianion, ... [摘要]  蛋白质磷酸化在细菌的信号转导中起着中心作用。然而,从其非磷酸化形式分离和检测磷酸化蛋白仍然是挑战性的。在这里我们描述了检测百日咳博德特氏菌响应调节剂BvgA的磷酸化的方法,其在天冬氨酸残基被磷酸化(Boulanger等人,2013)。该方法基于专有的加合物Phos-tag TM sup/TM,其是双核金属络​​合物,其与Zn 2+或Mn 2+反应, ,与磷酸二酯酶形成复合物,例如应答调节剂的磷酸化天冬氨酸(Barbieri和Stock,2008; Kinoshita和Kinoshita-Kikuta,2011)。对于体内检测,在4℃下在轻度甲酸中裂解百日咳细胞以使磷酸 - 天冬氨酸键的破坏最小化,并且通过包含Phos标签的电泳(SDS-PAGE)将磷酸化的BvgA从其非磷酸化形式分离> TM 。随后通过蛋白质印迹分析检测两种形式的BvgA。还容易实现在体外用乙酰磷酸盐处理后形成的磷酸化BvgA的水平的量化。因此,该技术允许容易地评估B中BvgA磷酸化的水平。百日咳和 。大肠杆菌在不同实验室条件下在体内或在不同反应条件下在体外磷酸化后(本研究部分由NIH的Intramural Research Programme支持, NIDDK)。

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