| Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method
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Author:
Date:
2017-06-20
[Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also named Thermofluor. We have shown that Thermofluor can also be applied to predict binding of substrates and inhibitors to a yeast α-glucosidase. The methods described here in detail were used in Viigand et al., 2016.
[摘要] α-葡糖苷酶（包括麦芽糖酶和异麦芽糖酶）是从一组α-葡糖苷底物释放葡萄糖的酶。 可以使用该特征来测定其催化活性，底物特异性和热稳定性。 蛋白质的热稳定性也可以使用高通量差示扫描荧光测定法（也称为Thermofluor）来测定。 我们已经表明，Thermofluor也可以应用于预测底物和抑制剂与酵母α-葡萄糖苷酶的结合。 这里详细描述的方法用于Viigand等人，2016。
【背景】麦芽糖酶（EC 3.2.1.20）和异麦芽糖酶（EC 3.2.1.10）是根据CAZy分类属于糖苷水解酶家族13的α-葡糖苷酶（Lombard等，2014）。甲基营养酵母多形汉酵母的麦芽糖酶MAL1是非选择性的，它将产生D-葡萄糖的麦芽糖和异麦芽糖状α-葡萄糖苷水解为反应产物之一。因此，麦芽糖酶对其底物的活性可以根据葡萄糖释放来确定。该工作描述的葡萄糖液色辅助方法可以快速方便地测定麦芽糖酶的活性，底物特异性和热稳定性。重要的是，这种基于活性的方法可以适用于产生葡萄糖作为反应产物的其它酶。高通量Thermofluor方法主要用于蛋白质晶体学测量（热）稳定性蛋白质（Boivin等，2013; Ericsson等，2006）。我们使用Thermofluor ...
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| 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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