| Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay
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Author:
Date:
2014-06-05
[Abstract] This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic efficiency of pseudokinases is rather poor or non existent, making it difficult to dissect the function of their nucleotide binding sites. Thermal denaturation-based methods have proven to be a powerful method for determining ligand binding capacity ...
[摘要] 该协议描述了通过基于荧光的热漂移测定（TSA）测量假激酶 - 配体相互作用的鲁棒技术。假激酶是激酶样蛋白，其最近作为信号转导的关键调节剂出现，因此可以代表一类新型的药物靶标。与激酶不同，假激酶的催化效率相当差或不存在，使得难以解析其核苷酸结合位点的功能。已证明基于热变性的方法是确定与纯化的假激酶的配体结合能力的有力方法，并且可以告知核苷酸结合位点的潜在可药物性。该测定利用了当荧光染料（在这种情况下为SYPROOrange）与当蛋白质经历热变性时暴露的疏水性贴片结合时产生的荧光变化。已知配体结合蛋白质增加其热稳定性，其通过在未配对蛋白质和配体蛋白质之间的热变性曲线中观察到的移动来反映。这一广义协议也可以适合于其他蛋白家族。此外，基于热变性的方法可用于鉴定可提高蛋白质稳定性的最佳缓冲液条件
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| Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra
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Author:
Date:
2013-11-20
[Abstract] Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, ...
[摘要] 木质素是沉积在二次增厚的植物细胞壁中的复杂酚类聚合物。聚合物主要衍生自当偶联时产生对羟基苯基，愈创木基和紫丁香基单元（分别为H，G和S单元）的三种主要单独的木质素：p - 香豆酰基，松柏基和芥子醇进入聚合物。结构单元的甲氧基化程度不同，它们的生物合成途径被超过10种酶催化。 HCT通过将苯丙素类导向松柏醇和芥子醇的生产来发挥关键作用。有趣的是，据报道，HCT涉及p - 香豆酰基莽草酸的芳环的3-羟基化的上游和下游途径（图1）（Hoffmann等， 2003; Hoffmann等人，2004; Vanholme等人，2013b）。这些特征突出了开发在植物中可靠地测量HCT活性的测定法的重要性。在这里，我们描述了基于UPLC-MS的方法，用于分析黑曲霉的木质部总蛋白提取物中的HCT活性，其可以适用于其他木本和草本植物物种。该方案最初在Vanholme等人（2013a）中描述。
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